Fused Pyrazine Compounds Useful for the Treatment of Degenerative and Inflammatory Diseases

ABSTRACT

Novel fused pyrazine compounds are disclosed that have a formula represented by the following: 
     
       
         
         
             
             
         
       
     
     The compounds may be prepared as pharmaceutical compositions, and may be used for the prevention and treatment of a variety of conditions in mammals including humans, including by way of non-limiting example, inflammation, rheumatoid arthritis and others.

RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 12/151,585, filed May 7, 2008, which claims the benefit under35 U.S.C. §119 of U.S. Provisional Application Ser. No. 60/928,600 filedMay 10, 2007, and Ser. No. 60/931,764, filed May 25, 2007, the contentsof all of which applications are hereby incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to a class of fused pyrazine compoundscapable of binding to the active site of a serine/threonine kinase, theexpression of which is involved in the pathway resulting in thedegradation of extra-cellular matrix (ECM), joint degeneration anddiseases involving such degradation and/or inflammation.

Diseases involving the degradation of extra-cellular matrix include, butare not limited to, psoriatic arthritis, juvenile arthritis, earlyarthritis, reactive arthritis, osteoarthritis, ankylosing spondylitis,osteoporosis, muskulo skeletal diseases like tendonitis and periodontaldisease, cancer metastasis, airway diseases (COPD, asthma), renal andliver fibrosis, cardio-vascular diseases like atherosclerosis and heartfailure, and neurological diseases like neuroinflammation and multiplesclerosis. Diseases involving primarily joint degeneration include, butare not limited to, psoriatic arthritis, juvenile arthritis, earlyarthritis, reactive arthritis, osteoarthritis, and ankylosingspondylitis.

Rheumatoid arthritis (RA) is a chronic joint degenerative disease,characterized by inflammation and destruction of the joint structures.When the disease is unchecked, it leads to substantial disability andpain due to loss of joint functionality and even premature death. Theaim of an RA therapy, therefore, is not to slow down the disease but toattain remission in order to stop the joint destruction. Besides theseverity of the disease outcome, the high prevalence of RA (˜0.8% of theadults are affected worldwide) means a high socio-economic impact. (Forreviews on RA, we refer to Smolen and Steiner (2003); Lee and Weinblatt(2001); Choy and Panayi (2001); O'Dell (2004) and Firestein (2003)).

Although it is widely accepted that RA is an auto-immune disease, thereis no consensus concerning the precise mechanisms driving the‘initiation stage’ of the disease. What is known is that the initialtrigger(s) does mediate, in a predisposed host, a cascade of events thatleads to the activation of various cell types (B-cells, T-cells,macrophages, fibroblasts, endothelial cells, dendritic cells andothers). Concomitantly, an increased production of various cytokines isobserved in the joints and tissues surrounding the joint (e.g. TNF-α,IL-6, IL-1, IL-15, IL-18 and others). When the disease progresses, thecellular activation and cytokine production cascade becomesself-perpetuating. At this early stage, the destruction of jointstructures is already very clear at this early stage. Thirty percent ofthe patients have radiographic evidence of bony erosions at the time ofdiagnosis and this proportion increases to 60 percent after two years.

Histological analysis of the joints of RA patients clearly evidences themechanisms involved in the RA-associated degradative processes. Thisanalysis shows that the main effector responsible for RA-associatedjoint degradation is the pannus, where the synovial fibroblast, byproducing diverse proteolytic enzymes, is the prime driver of cartilageand bone erosion. A joint classically contains two adjacent bones thatarticulate on a cartilage layer and are surrounded by the synovialmembrane and joint capsule. In the advanced RA patient, the synovium ofjoint increases in size to form the pannus, due to the proliferation ofthe synovial fibroblasts and the infiltration of mononuclear cells suchas T-cells, B-cells, monocytes, macrophages and neutrophils. The pannusmediates the degradation of the adjacent cartilage, leading to thenarrowing of the joint space, and has the potential to invade adjacentbone and cartilage. As bone and cartilage tissues are composed mainly ofcollagen type I or II, respectively, the pannus destructive and invasiveproperties are mediated by the secretion of collagenolytic proteases,principally the matrix metallo proteinases (MMPs). The erosion of thebone under and adjacent to the cartilage is also part of the RA process,and results principally from the presence of osteoclasts at theinterface of bone and pannus. Osteoclasts are multinucleated cells that,upon adhesion to the bone tissue, form a closed compartment, withinwhich the osteoclasts secrete proteases (Cathepsin K, MMP9) that degradethe bone tissue. The osteoclast population in the joint is abnormallyincreased by osteoblast formation from precursor cells induced by thesecretion of the receptor activator of NFκB ligand (RANKL) by activatedSFs and T-cells.

Various collagen types have a key role in defining the stability of theextracellular matrix (ECM). Collagen type I and collagen type II, forexample, are the main components of bone and cartilage, respectively.Collagen proteins typically organise into multimeric structures referredto as collagen fibrils. Native collagen fibrils are very resistant toproteolytic cleavage. Only a few types of ECM-degrading proteins havebeen reported to have the capacity to degrade native collagen: MMPs andCathepsins. Among the Cathepsins, cathepsin K, which is active mainly inosteoclasts, is the best characterised. Among the MMPs, MMP1, MMP2, MMP8MMP13 and MMP14 are known to have collagenolytic properties. Thecorrelation between an increased expression of MMP1 by synovialfibroblasts (SFs) and the progression of the arthritic disease iswell-established and is predictive for joint erosive processes (Cunnaneet al., 2001). In the context of RA, therefore, MMP1 represents a highlyrelevant collagen degrading protein. In vitro, the treatment of culturedSFs with cytokines relevant in the RA pathology (e.g. TNF-α and IL1β)will increase the expression of MMP1 by these cells (Andreakos et al.,2003). Monitoring the levels of MMP1 expressed by SFs therefore is arelevant readout in the field of RA as it is indicative for theactivation of SFs towards an erosive phenotype that, in vivo, isresponsible for cartilage degradation. Inhibition of the MMP1 expressionby SFs represents a valuable therapeutic approach towards the treatmentof RA.

The activity of the ECM-degrading proteins can also be causative orcorrelate with the progression of various diseases different from RA, ase.g. other diseases that involve the degradation of the joints. Thesediseases include, but are not limited to, psoriatic arthritis, juvenilearthritis, early arthritis, reactive arthritis, osteoarthritis, andankylosing spondylitis. Other diseases that may be treatable withcompounds identified according to the present invention and using thetargets involved in the expression of MMPs as described herein areosteoporosis, muscular skeletal diseases like tendonitis and periodontaldisease (Gapski et al., 2004), cancer metastasis (Coussens et al.,2002), airway diseases (COPD, asthma) (Suzuki et al., 2004), lung, renalfibrosis (Schanstra et al., 2002), liver fibrosis associated withchronic hepatitis C (Reiff et al., 2005), cardio-vascular diseases likeatherosclerosis and heart failure (Creemers et al., 2001), andneurological diseases like neuroinflammation and multiple sclerosis(Rosenberg, 2002). Patients suffering from such diseases may benefitfrom stabilizing the ECM (by protecting it from degradation).

The 471-amino acid serine/threonine kinase identified asMitogen-Activated Protein Kinase-Activated Protein Kinase 5 (MAPKAPK5 orPRAK) is expressed in a wide panel of tissues. The protein contains itscatalytic domain at the N-terminal end and both a nuclear localizationsignal (NLS) and nuclear export signal (NES) at its C-terminal end.Endogenous MAPKAPK5 is predominantly present in the cytoplasm, butstress or cytokine activation of the cells mediates its translocationinto the nucleus (New et al., 2003). This event is dependent onphosphorylation of MAPKAPK5. Thr182 is the regulatory phosphorylationsite of MAPKAPK5. Although the p38α kinase is able to phosphorylateMAPKAPK5 in an overexpression setting, experiments with endogenousMAPKAPK5 do not support this hypothesis (Shi et al., 2003). MAPKAPK5knock-out mice have been generated that are viable and fertile. Thephenotype of these mice is quite different from that of mice deficientfor MAPKAPK2, a MAPKAPK5 related kinase that is regulated by p38α (Shiet al., 2003). This indicates that the function of each protein isdistinct and that neither kinase can compensate for the other'sactivity. Taken together, MAPKAPK5 and MAPKAPK2 represent distincttargets with a non-redundant role. MAPK6 (also referred to as ERK3) hasrecently been identified as a physiologically relevant substrate forMAPKAPK5, defining a novel signal transduction pathway (Seternes et al.,2004).

BACKGROUND OF THE INVENTION

NSAIDS (Non-steroidal anti-inflammatory drugs) are used to reduce thepain associated with RA and improve life quality of the patients. Thesedrugs will not, however, put a brake on the RA-associated jointdestruction.

Corticosteroids were found to decrease the progression of RA as detectedradiographically and are used at low doses to treat part of the RApatients (30 to 60%). Serious side effects, however, are associated withlong corticosteroid use (Skin thinning, osteoporosis, cataracts,hypertension and hyperlipidemia).

Synthetic DMARDs (Disease-Modifying Anti-Rheumatic Drugs) (e.g.methotrexate, leflunomide, sulfasalazine) mainly tackle theimmuno-inflammatory component of RA. As a main disadvantage, these drugsonly have a limited efficacy (joint destruction is only slowed down butnot blocked by DMARDs such that disease progression in the long termcontinues). The lack of efficacy is indicated by the fact that, onaverage, only 30% of the patients achieve an ACR40 score after 24 monthstreatment with methotrexate. This means that, according to the AmericanCollege of Rheumatology, only 30% of the patients do achieve a 50%improvement of their symptoms (O'Dell et al., 1996). In addition, theprecise mechanism of action of DMARDs is often unclear.

Biological DMARDs (Infliximab, Etanercept, Adalimumab, Rituximab;CTLA4-Ig) are therapeutic proteins that do inactivate cytokines (e.g.TNF-α) or cells (e.g. T-cells or B-cells) that have an important role inthe RA pathophysiology (Kremer et al., 2003; Edwards et al., 2004).Although the TNF-α-blockers (Infliximab, Etanercept, Adalimumab) andmethotrexate combination therapy is the most effective RA treatmentcurrently available, it is striking that even this therapy only achievesa 50% improvement (ACR40) in disease symptoms in 50-60% of patientsafter 12 months therapy (St Clair et al., 2004). Some adverse eventswarnings for anti-TNF-α drugs exist, shedding a light on the sideeffects associated to this type of drugs. Increased risk for infections(tuberculosis), hematologic events and demyelinating disorders have beendescribed for the TNF-α blockers (see also Gomez-Reino et al., 2003).Besides the serious side effects, the TNF-α blockers also share thegeneral disadvantages of the biological class of therapeutics, which arethe unpleasant way of administration (frequent injections accompanied byinfusion site reactions) and the high production cost. Newer agents inlate development phase target T-cell co-stimulatory molecules andB-cells. The efficacy of these agents is expected to be similar to thatof the TNF-α blockers. The fact that a variety of targeted therapieshave similar but limited efficacies, suggests that there is amultiplicity of pathogenic factors for RA. This is also indicative forthe deficiencies in our understanding of pathogenic events relevant toRA.

The current therapies for RA are not satisfactory due to a limitedefficacy (No adequate therapy exists for 30% of the patients). Thiscalls for additional strategies to achieve remission. Remission isrequired since residual disease bears the risk of progressive jointdamage and thus progressive disability. Inhibiting theimmuno-inflammatory component of the RA disease, which represents themain target of drugs currently used for RA treatment, does not result ina blockade of joint degradation, the major hallmark of the disease.

US 2005/0009832 describes substituted imidazo[1,2-a]pyrazine-8-yl-aminesas modulators of protein kinases, including MAPKAPK5. WO02/056888describes inhibitors of MAPKAPK5 as TNF modulators able to regulate theexpression of certain cytokines. Neither of these prior art referencesdiscloses any compound within the scope of the class of compoundsdescribed herein below.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that MAPKAPK5 functionsin the pathway that results in the expression of MMP1, and thatinhibitors of MAPKAPK5 activity, such as the compounds of the presentinvention, are useful for the treatment of diseases involving theabnormally high expression of MMP activity.

The compounds of the present invention may be described generally asfused pyrazines, in particular imidazo[1,2-a]pyrazine-8-yl-amines and[1.2.4]triazolo[1,5-a]pyrazine-8-yl-amines substituted in the 5-positionby an aromatic group capable of donating electrons to, and an 8-aminosubstituent capable of accepting electrons from, the fused pyrazine,i.e. the imidazo[1,2.a]pyrazine or the [1.2.4]triazolo[1,5-a]pyrazinering. In particular, the 5-substituent group is characterized as havinga hydrogen bond donor-acceptor functionality, whereas the substituent onthe 8-amino group must be sufficiently electron-withdrawing to polarisethe N—H bond of the 8-substituent, or alternatively, the 8-NH group iscapable of participating in pi-conjugation with the substituent group onthe 8-NH group.

The compounds of the present invention may show less toxicity, goodabsorption, good half-life, good solubility, low protein bindingaffinity, less drug-drug interaction, and good metabolic stability.

More particularly, the present invention relates to compounds accordingto formula (I):

wherein

Z is CH or N; R¹ is H, or substituted or unsubstituted C₁-C₆ alkyl; R²is H, C₁-C₆ alkyl, C₃-C₇ cycloalkyl or C₃-C₇ cycloalkyl-C₁-C₆ alkyl,optionally substituted with one or more of F and Cl; R⁸ is substitutedor unsubstituted heteroaryl; R⁹ is selected from substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl; or apharmaceutically acceptable salt, solvate or prodrug thereof; andstereoisomers, isotopic variants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, R¹ is H.

In one embodiment, with respect to compounds of formula I, R² is H.

In one embodiment, with respect to compounds of formula I, R⁸ issubstituted or unsubstituted 5-membered heteroaryl.

In another embodiment, with respect to compounds of formula I, R⁸ isselected from substituted or unsubstituted pyrrolyl, furanyl, thienyl,pyrazolyl, oxazolyl, imidazolyl, thiazolyl, 1,3,4-thiadiazolyl, and1,2,4-thiadiazolyl.

In another embodiment, with respect to compounds of formula I, R⁸ isselected from substituted pyrrolyl, furanyl, thienyl, pyrazolyl,oxazolyl, imidazolyl, thiazolyl, 1,3,4-thiadiazolyl, and1,2,4-thiadiazolyl and the substitution is selected from alkyl,substituted alkyl, carbamoyl, hydroxyalkyl, haloalkyl, alkoxy, cyano,amino, sulfo, aryl, heterocycloalkyl, aralkyl, heterocycloalkyl andheteroaryl.

In another embodiment, with respect to compounds of formula I, R⁹ isselected from substituted or unsubstituted phenyl.

In another embodiment, with respect to compounds of formula I, R⁹ isselected from substituted or unsubstituted pyridyl.

In another embodiment, with respect to compounds of formula I, R⁹ isselected from substituted or unsubstituted phenyl, indolyl, isoinolyl,pyrrolyl, furanyl, thienyl, pyrazolyl, oxazolyl, and thiazolyl.

In a further aspect, the present invention provides pharmaceuticalcompositions comprising a fused pyrazine compound of the invention, anda pharmaceutical carrier, excipient or diluent. In this aspect of theinvention, the pharmaceutical composition can comprise one or more ofthe compounds described herein. Moreover, the compounds of the presentinvention useful in the pharmaceutical compositions and treatmentmethods disclosed herein, are all pharmaceutically acceptable asprepared and used.

Another aspect of this invention relates to the use of a compound of theinvention in a therapeutic method, a pharmaceutical composition, and themanufacture of such composition, useful for the treatment of diseasesinvolving inflammation, collagen degradation, and in particular,diseases characteristic of abnormal matrix metallo protease (MMP1)and/or Mitogen-Activated Protein-Kinase Activated Protein Kinase 5(MAPKAPK5) activity, of which rheumatoid arthritis (RA) is a particularsuch disease. This invention also relates to processes for thepreparation of the present compounds.

Another aspect of this invention relates to a compound of the inventionfor use in the treatment of diseases involving inflammation, collagendegradation, and in particular, diseases characteristic of abnormalmatrix metallo protease (MMP1) and/or Mitogen-Activated Protein-KinaseActivated Protein Kinase 5 (MAPKAPK5) activity, of which rheumatoidarthritis (RA) is a particular such disease.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing detailed description,considered in conjunction with the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. This diagram shows the striking histological differences betweena healthy joint and that of a RA patient.

FIG. 2. This chart shows the increased expression of MMP1 in synovialfibroblasts triggered with cytokines involved in rheumatoid arthritispathology.

FIG. 3. This graph shows the dose-dependent inhibition of the“TNF-α-based trigger”-induced expression of MMP1 by SFs by a knownanti-inflammatory compound.

FIG. 4. This gel shows the reduction, at the protein level, of theexpression of MAPKAPK5 in SFs by infection of the cells with Ad-siRNAvirus targeting MAPKAPK5.

FIG. 5. This chart shows the reduction of ‘complex trigger’ inducedlevels of MMP1 expression by SFs by an Ad-siRNA virus targetingMAPKAPK5.

DETAILED DESCRIPTION OF THE INVENTION

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description andintended scope of the present invention.

When describing the invention, which may include compounds,pharmaceutical compositions containing such compounds and methods ofusing such compounds and compositions, the following terms, if present,have the following meanings unless otherwise indicated. It should alsobe understood that when described herein any of the moieties definedforth below may be substituted with a variety of substituents, and thatthe respective definitions are intended to include such substitutedmoieties within their scope as set out below. Unless otherwise stated,the term “substituted” is to be defined as set out below. It should befurther understood that the terms “groups” and “radicals” can beconsidered interchangeable when used herein.

The articles “a” and “an” may be used herein to refer to one or to morethan one (i.e. at least one) of the grammatical objects of the article.By way of example “an analogue” means one analogue or more than oneanalogue.

‘Acyl’ refers to a radical —C(O)R²⁰, where R²⁰ is hydrogen, alkyl,cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroarylor heteroarylalkyl as defined herein. Representative examples include,but are not limited to, formyl, acetyl, cylcohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl and benzylcarbonyl. Exemplary ‘acyl’groups are —C(O)H, —C(O)—C₁-C₈ alkyl, —C(O)—(CH₂)_(t)(C₆-C₁₀ aryl),—C(O)—(CH₂)_(t)(C₅-C₁₀ heteroaryl), —C(O)—(CH₂)_(t)(C₃-C₁₀ cycloalkyl),and —C(O)—(CH₂)_(t)(C₅-C₁₀ heterocycloalkyl), wherein t is an integerfrom 0 to 4 and any aryl, heteroaryl, cycloalkyl or heterocycloalkylgroups present, may themselves be substituted by C₁-C₄ alkyl, halo,C₁-C₄ alkoxy, C₁₋₄ haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy orhydroxy.

‘Acylamino’ refers to a radical —NR²¹C(O)R²², where R²¹ is hydrogen,alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl,heteroaryl or heteroarylalkyl and R²² is hydrogen, alkyl, alkoxy,cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroarylor heteroarylalkyl, as defined herein. Representative examples include,but are not limited to, formylamino, acetylamino,cyclohexylcarbonylamino, cyclohexylmethyl-carbonylamino, benzoylaminoand benzylcarbonylamino. Exemplary ‘acylamino’ groups are—NR²¹C(O)—C₁-C₈ alkyl, —NR²¹C(O)—(CH₂)_(t)(C₆-C₁₀ aryl),—NR²¹C(O)—(CH₂)_(t)(C₅-C₁₀ heteroaryl), —NR²¹C(O)—(CH₂)_(t)(C₃-C₁₀cycloalkyl), and —NR²¹C(O)(CH₂)_(t)(C₅-C₁₀ heterocycloalkyl), wherein tis an integer from 0 to 4, each R²¹ independently represents H or C₁-C₆alkyl and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groupspresent, may themselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄alkoxy, C₁-C₄ haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy orhydroxy.

‘Acyloxy’ refers to the group —OC(O)R²³ where R²³ is hydrogen, alkyl,aryl or cycloalkyl. Exemplary ‘acyloxy’ groups are OC(O)—C₁-C₈ alkyl,—OC(O)—(CH₂)_(t)(C₆-C₁₀ aryl), —OC(O)—(CH₂)_(t)(C₅-C₁₀ heteroaryl),—OC(O)—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and —OC(O)—(CH₂)_(t)(C₅-C₁₀heterocycloalkyl), wherein t is an integer from 0 to 4 and any aryl,heteroaryl, cycloalkyl or heterocycloalkyl groups present, maythemselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄ alkoxy, C₁₋₄haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy or hydroxy.

‘Alkoxy’ refers to the group —OR²⁴ where R²⁴ is alkyl. Particular alkoxygroups are, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy and 1,2-dimethylbutoxy.Particular alkoxy groups are lower alkoxy, i.e. with between 1 and 6carbon atoms.

‘Substituted alkoxy’ refers to an alkoxy group substituted with one ormore of those groups recited in the definition of “substituted” herein,and particularly refers to an alkoxy group having 1 or moresubstituents, for instance from 1 to 5 substituents, and particularlyfrom 1 to 3 substituents, in particular 1 substituent, selected from thegroup consisting of acyl, acylamino, acyloxy, alkoxy, substitutedalkoxy, alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,heteroaryl, hydroxyl, keto, nitro, thioalkoxy, substituted thioalkoxy,thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— andaryl-S(O)₂—. Exemplary ‘substituted alkoxy’ groups are—O—(CH₂)_(t)(C₆-C₁₀ aryl), —O—(CH₂)_(t)(C₅-C₁₀ heteroaryl),—O—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and —O—(CH₂)_(t)(C₅-C₁₀heterocycloalkyl), wherein t is an integer from 0 to 4 and any aryl,heteroaryl, cycloalkyl or heterocycloalkyl groups present, maythemselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄ alkoxy, C₁₋₄haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy or hydroxy.

‘Alkoxycarbonylamino’ refers to the group —NR²⁵C(O)OR²⁶, where R²⁵ ishydrogen, alkyl, aryl or cycloalkyl, and R²⁶ is alkyl or cycloalkyl.

‘Alkyl’ means straight or branched aliphatic hydrocarbon having 1 toabout 20 carbon atoms. Particular alkyl has 1 to about 12 carbon atoms.More particular is lower alkyl which has 1 to 6 carbon atoms. Mostparticular are groups such as methyl, ethyl and propyl. Branched meansthat one or more lower alkyl groups such as methyl, ethyl or propyl isattached to a linear alkyl chain. The term C₁-C₆ alkyl includes bothbranched and straight chain groups, exemplary straight chain groupsinclude ethyl, propyl, butyl, exemplary branched chain groups includeisopropyl, isoamyl, and the like.

‘Substituted alkyl’ refers to an alkyl group substituted with one ormore of those groups recited in the definition of “substituted” herein,and particularly refers to an alkyl group having 1 or more substituents,for instance from 1 to 5 substituents, and particularly from 1 to 3substituents, in particular 1 substituent, selected from the groupconsisting of acyl, acylamino, acyloxy, alkoxy, substituted alkoxy,alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy,azido, carboxyl, cyano, cycloalkyl, substituted cycloalkyl, halogen,hydroxyl, heteroaryl, keto, nitro, thioalkoxy, substituted thioalkoxy,thioaryloxy, thioketo, thiol, alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂—, andaryl-S(O)₂—. In a particular embodiment ‘substituted alkyl’ refers to aC₁-C₆ alkyl group substituted with halo, cyano, nitro, trifluoromethyl,trifluoromethoxy, azido, —NR′″SO₂R″, —SO₂NR″R′″, —C(O)R″, —C(O)OR″,—OC(O)R″, —NR′″C(O)R″, —C(O)NR″R′″, —NR″R′″, or —(CR′″R″″)_(m)OR′″;wherein each R″ is independently selected from H, C₁-C₈ alkyl,—(CH₂)_(t)(C₆-C₁₀ aryl), —(CH₂)_(t)(C₅-C₁₀ heteroaryl),—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and —(CH₂)_(t)(C₅-C₁₀ heterocycloalkyl),wherein t is an integer from 0 to 4 and any aryl, heteroaryl, cycloalkylor heterocycloalkyl groups present, may themselves be substituted byC₁-C₄ alkyl, halo, C₁-C₄ alkoxy, C₁₋₄ haloalkyl, C₁-C₄ hydroxyalkyl, orC₁-C₄ haloalkoxy or hydroxy. Each of R′″ and R″″ independentlyrepresents H or C₁-C₆ alkyl.

‘Alkylene’ refers to divalent alkene radical groups having 1 to 11carbon atoms and more particularly 1 to 6 carbon atoms which can bestraight-chained or branched. This term is exemplified by groups such asmethylene (—CH₂—), ethylene (—CH₂CH₂—) and the propylene isomers (e.g.,—CH₂CH₂CH₂— and —CH(CH₃)CH₂—).

‘Substituted alkylene’ refers to an alkylene group substituted with oneor more of those groups recited in the definition of ‘substituted’herein, and particularly refers to an alkylene group having 1 or moresubstituents, for instance from 1 to 5 substituents, and particularlyfrom 1 to 3 substituents, in particular I substituent.

‘Alkenyl’ refers to monovalent olefinically (unsaturated) hydrocarbongroups preferably having 2 to 11 carbon atoms, particularly, from 2 to 8carbon atoms, and more particularly, from 2 to 6 carbon atoms, which canbe straight-chained or branched and having at least 1 and particularlyfrom 1 to 2 sites of olefinic unsaturation. Particular alkenyl groupsinclude ethenyl (—CH═CH₂), n-propenyl (—CH₂CH═CH₂), isopropenyl(—C(CH₃)═CH₂), vinyl and substituted vinyl.

‘Substituted alkenyl’ refers to an alkenyl group substituted with one ormore of those groups recited in the definition of ‘substituted’ herein,and particularly refers to an alkenyl group having 1 or moresubstituents, for instance from 1 to 5 substituents, and particularlyfrom 1 to 3 substituents, in particular 1 substituent.

‘Alkenylene’ refers to divalent olefinically (unsaturated) hydrocarbongroups particularly having up to about 11 carbon atoms and moreparticularly 2 to 6 carbon atoms which can be straight-chained orbranched and having at least 1 and particularly from 1 to 2 sites ofolefinic unsaturation. This term is exemplified by groups such asethenylene (—CH═CH—) and the propenylene isomers (e.g., —CH═CHCH₂— and—C(CH₃)═CH— and —CH═C(CH₃)—).

‘Alkynyl’ refers to acetylenically or alkynically (unsaturated)hydrocarbon groups particularly having 2 to 11 carbon atoms and moreparticularly 2 to 6 carbon atoms which can be straight-chained orbranched and having at least 1 and particularly from 1 to 2 sites ofalkynyl unsaturation. Particular non-limiting examples of alkynyl groupsinclude acetylenic, ethynyl (—C≡CH), and propargyl (—CH₂C≡CH).

‘Substituted alkynyl’ refers to an alkynyl group substituted with one ormore of those groups recited in the definition of “substituted” herein,and particularly refers to an alkynyl group having 1 or moresubstituents, for instance from 1 to 5 substituents, and particularlyfrom 1 to 3 substituents, in particular 1 substituent.

‘Alkanoyl’ or ‘acyl’ as used herein refers to the group R²⁷—C(O)—, whereR²⁷ is hydrogen or alkyl as defined above.

‘Aryl’ refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. In particular aryl refers to an aromatic ringstructure, mono-cyclic or poly-cyclic that includes from 5 to 12 ringmembers, more usually 5 to 10. Where the aryl group is a monocyclic ringsystem it preferentially contains 6 carbon atoms. Typical aryl groupsinclude, but are not limited to, groups derived from aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexylene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene;pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene andtrinaphthalene. Particularly aryl groups include phenyl, naphthyl,indenyl, and tetrahydronaphthyl. The term ‘aryl’ includes ‘bicycloaryl’as defined below.

‘Bicycloaryl’ refers to a monovalent aromatic hydrocarbon group derivedby the removal of one hydrogen atom from a single carbon atom of aparent bicycloaromatic ring system. Typical bicycloaryl groups include,but are not limited to, groups derived from indane, indene, naphthaleneand tetrahydronaphthalene. Particularly, an aryl group comprises from 8to 11 carbon atoms.

‘Substituted Aryl’ refers to an aryl group substituted with one or moreof those groups recited in the definition of ‘substituted’ herein, andparticularly refers to an aryl group that may optionally be substitutedwith 1 or more substituents, for instance from 1 to 5 substituents,particularly 1 to 3 substituents, in particular 1 substituent.

‘Fused Aryl’ refers to an aryl having two of its ring carbon in commonwith a second aryl ring or with an aliphatic ring.

‘Alkaryl’ or ‘arylalkyl’ refers to an aryl group, as defined above,substituted with one or more alkyl groups, as defined above.

‘Substituted Aralkyl’ or ‘substituted arylalkyl’ refers to an alkylgroup, as defined above, substituted with one or more aryl groups, asdefined above.

‘Aryloxy’ refers to —O-aryl groups wherein ‘aryl’ is as defined above.

‘Alkylamino’ refers to the group alkyl-NR²⁸R²⁹, wherein each of R²⁸ andR²⁹ are independently selected from hydrogen and alkyl.

‘Arylamino’ refers to the group aryl-NR³⁰R³¹, wherein each of R³⁰ andR³¹ are independently selected from hydrogen, aryl and heteroaryl.

‘Alkoxyamino’ refers to a radical —N(H)OR³² where R³² represents analkyl or cycloalkyl group as defined herein.

‘Alkoxycarbonyl’ refers to a radical —C(O)-alkoxy where alkoxy is asdefined herein. Exemplary “alkoxycarbonyl” groups are C(O)O—C₁-C₈ alkyl,—C(O)O—(CH₂)_(t)(C₆-C₁₀ aryl), —C(O)O—(CH₂)_(t)(C₅-C₁₃ heteroaryl),—C(O)O—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and —C(O)O—(CH₂)_(t)(C₅-C₁₀heterocycloalkyl), wherein t is an integer from 0 to 4 and any aryl,heteroaryl, cycloalkyl or heterocycloalkyl groups present, maythemselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄ alkoxy, C₁₋₄haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy or hydroxy.

‘Alkylarylamino’ refers to a radical —NR³³R³⁴ where R³³ represents analkyl or cycloalkyl group and R³⁴ is an aryl as defined herein.

‘Alkylsulfonyl’ refers to a radical —S(O)₂R³⁵ where R³⁵ is an alkyl orcycloalkyl group as defined herein. Representative examples include, butare not limited to, methylsulfonyl, ethylsulfonyl, propylsulfonyl andbutylsulfonyl.

‘Alkylsulfinyl’ refers to a radical —S(O)R³⁵ where R³⁵ is an alkyl orcycloalkyl group as defined herein. Representative examples include, butare not limited to, methylsulfinyl, ethylsulfinyl, propylsulfinyl andbutylsulfinyl.

‘Alkylthio’ refers to a radical —SR³⁵ where R³⁵ is an alkyl orcycloalkyl group as defined herein that may be optionally substituted asdefined herein. Representative examples include, but are not limited to,methylthio, ethylthio, propylthio and butylthio. Exemplary ‘alkylthio’groups are S—C₁-C₈ alkyl, —S—(CH₂)_(t)(C₆-C₁₀ aryl), —S—(CH₂)_(t)(C₅-C₁₀heteroaryl), —S—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and —S—(CH₂)_(t)(C₅-C₁₀heterocycloalkyl), wherein t is an integer from 0 to 4 and any aryl,heteroaryl, cycloalkyl or heterocycloalkyl groups present, maythemselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄ alkoxy, C₁-₄haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy or hydroxy.

‘Amino’ refers to the radical —NH₂.

‘Substituted amino’ refers to an amino group substituted with one ormore of those groups recited in the definition of ‘substituted’ herein,and particularly refers to the group —N(R³⁶)₂ where each R³⁶ isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, cycloalkyl, substituted cycloalkyl, and where both Rgroups are joined to form an alkylene group. When both R groups arehydrogen, —N(R³⁶)₂ is an amino group. Exemplary ‘substituted amino’groups are —NR³⁶—C₁-C₈ alkyl, —NR³⁶—(CH₂)_(t)(C₆-C₁₀, aryl),—NR³⁶—(CH₂)_(t)(C₅-C₁₀ heteroaryl), —NR³⁶—(CH₂)_(t)(C₃-C₁₀, cycloalkyl),and —NR³⁶—(CH₂)_(t)(C₅-C₁₀ heterocycloalkyl), wherein t is an integerfrom 0 to 4, each R³⁶ independently represents H or C₁-C₆ alkyl and anyaryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, maythemselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄ alkoxy, C₁₋₄haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy or hydroxy.

‘Aminocarbonyl’ refers to the group —C(O)NR³⁷R³⁷ where each R³⁷ isindependently hydrogen, alkyl, aryl and cycloalkyl, or where the R³⁷groups are joined to form an alkylene group.

‘Aminocarbonylamino’ refers to the group —NR³⁸C(O)NR³⁸R³⁸ where each R³⁸is independently hydrogen, alkyl, aryl or cycloalkyl, or where two Rgroups are joined to form an alkylene group.

‘Aminocarbonyloxy’ refers to the group —OC(O)NR³⁹R³⁹ where each R³⁹ isindependently hydrogen, alkyl, aryl or cycloalkyl, or where the R groupsare joined to form an alkylene group.

‘Arylalkyloxy’ refers to an —O-arylalkyl radical where arylalkyl is asdefined herein.

‘Arylamino’ means a radical —NHR⁴⁰ where R⁴⁰ represents an aryl group asdefined herein.

‘Aryloxycarbonyl’ refers to a radical —C(O)—O-aryl where aryl is asdefined herein.

‘Arylsulfonyl’ refers to a radical —S(O)₂R⁴¹ where R⁴¹ is an aryl orheteroaryl group as defined herein.

‘Azido’ refers to the radical —N₃.

‘Carbamoyl’ refers to the radical —C(O)N(R⁴²)₂ where each R⁴² group isindependently hydrogen, alkyl, cycloalkyl or aryl, as defined herein,which may be optionally substituted as defined herein. A particularcarbamoyl group is —C(O)NH₂. Exemplary ‘carbamoyl’ groups are—C(O)NR⁴²—C₁-C₈ alkyl, —C(O)NR⁴²—(CH₂)_(t)(C₆-C₁₀ aryl),—C(O)NR⁴²—(CH₂)_(t)(C₅-C₁₀, heteroaryl), —C(O)NR⁴²—(CH₂)_(t)(C₃-C₁₀cycloalkyl), and —C(O)NR⁴²—(CH₂)_(t)(C₅-C₁₀ heterocycloalkyl), wherein tis an integer from 0 to 4, each R⁴² independently represents H or C₁-C₆alkyl and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groupspresent, may themselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄alkoxy, C₁₋₄ haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy orhydroxy.

‘Carboxy’ refers to the radical —C(O)OH.

‘Carboxyamino’ refers to the radical —N(H)C(O)OH.

‘Cycloalkyl’ refers to cyclic non-aromatic hydrocarbyl groups havingfrom 4 to about 7 carbon atoms and having a single cyclic ring, whichoptionally can be substituted with from 1 to 3 alkyl groups. Suchcycloalkyl groups include, by way of example, single ring structuressuch as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl,1-methylcyclopropyl, 2-methylcyclopentyl and 2-methylcyclooctyl.

‘Substituted cycloalkyl’ refers to a cycloalkyl group substituted withone or more of those groups recited in the definition of ‘substituted’herein, and particularly refers to a cycloalkyl group having 1 or moresubstituents, for instance from 1 to 5 substituents, and particularlyfrom 1 to 3 substituents, in particular 1 substituent

‘Cycloalkoxy’ refers to the group —OR⁴³ where R⁴³ is cycloalkyl. Suchcycloalkoxy groups include, by way of example, cyclopentoxy andcyclohexoxy.

‘Cycloalkenyl’ refers to cyclic hydrocarbyl groups having from 3 to 10carbon atoms and having a single cyclic ring or multiple condensedrings, including fused and bridged ring systems and having at least oneand particularly from 1 to 2 sites of olefinic unsaturation. Suchcycloalkenyl groups include, by way of example, single ring structuressuch as cyclohexenyl, cyclopentenyl and cyclopropenyl.

‘Substituted cycloalkenyl’ refers to a cycloalkenyl group substitutedwith one or more of those groups recited in the definition of“substituted” herein, and particularly refers to a cycloalkenyl grouphaving 1 or more substituents, for instance from 1 to 5 substituents,and particularly from 1 to 3 substituents, in particular 1 substituent

‘Fused Cycloalkenyl’ refers to a cycloalkenyl having two of its ringcarbon atoms in common with a second aliphatic or aromatic ring andhaving its olefinic unsaturation located to impart aromaticity to thecycloalkenyl ring.

‘Cyanato’ refers to the radical —OCN.

‘Cyano’ refers to the radical —CN.

‘Dialkylamino’ means a radical —NR⁴⁴R⁴⁵ where R⁴⁴ and R⁴⁵ independentlyrepresent an alkyl, substituted alkyl, aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitutedcycloheteroalkyl, heteroaryl, or substituted heteroaryl group as definedherein.

‘Ethenyl’ refers to substituted or unsubstituted —(C═C)—.

‘Ethylene’ refers to substituted or unsubstituted —(C—C)—.

‘Ethynyl’ refers to —(C≡C)—.

‘Halo’ or ‘halogen’ refers to fluoro (F), chloro (Cl), bromo (Br) andiodo (I). Preferred halo groups are either fluoro or chloro.

‘Hydrogen’ means in the context of a substituent that —H is present atthe compound position and also includes its isotope, deuterium.

‘Hydroxy’ refers to the radical —OH.

‘Nitro’ refers to the radical —NO₂.

‘Substituted’ refers to a group in which one or more hydrogen atoms areeach independently replaced with the same or different substituent(s).Typical substituents are selected from the group consisting of: —X,—R⁴⁶, —O⁺, ═O, —OR⁴⁶, —SR⁴⁶, —S⁻, ═S, —NR⁴⁶R⁴⁷, ═NR⁴⁶, —CX₃, —CF₃, —CN,—OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R⁴⁶,—OS(O₂)O⁻, —OS(O)₂R⁴⁶, —P(O)(O⁻)₂, —P(O)(OR⁴⁶)(O⁻), —OP(O)(OR⁴⁶)(OR⁴⁷),—C(O)R⁴⁶, —C(S)R⁴⁶, —C(O)OR⁴⁶, —C(O)NR⁴⁶R⁴⁷, —C(O)O⁻, —C(S)OR⁴⁶,—NR⁴⁸C(O)NR⁴⁶R⁴⁷, NR⁴⁸C(S)NR⁴⁶R⁴⁷, —NR⁴⁹C(NR⁴⁸)NR⁴⁶R⁴⁷ and—C(NR⁴⁸)NR⁴⁶R⁴⁷, where each X is independently a halogen; each R⁴⁶, R⁴⁷,R⁴⁸ and R⁴⁹ are independently hydrogen, alkyl, substituted alkyl, aryl,substituted alkyl, arylalkyl, substituted alkyl, cycloalkyl, substitutedalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, —NR⁵⁰R⁵¹, —C(O)R⁵⁰ or—S(O)₂R⁵⁰ or optionally R⁵⁰ and R⁵¹ together with the atom to which theyare both attached form a cycloheteroalkyl or substitutedcycloheteroalkyl ring; and R⁵⁰ and R⁵¹ are independently hydrogen,alkyl, substituted alkyl, aryl, substituted alkyl, arylalkyl,substituted alkyl, cycloalkyl, substituted alkyl, cycloheteroalkyl,substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl or substitutedheteroarylalkyl. In a particular embodiment, substituted groups aresubstituted with one or more substituents, particularly with 1 to 3substituents, in particular with one substituent group. In a furtherparticular embodiment the substituent group or groups are selected fromhalo, cyano, nitro, trifluoromethyl, trifluoromethoxy, azido,—NR′″SO₂R″, —SO₂NR″R′″, —C(O)R″, —C(O)OR″, —OC(O)R″, —NR′″C(O)R″,—C(O)NR″R′″, —NR″R′″, —(CR′″R′″)_(m)OR′″, wherein, each R″ isindependently selected from H, C₁-C₈ alkyl, —(CH₂)_(t)(C₆-C₁₀ aryl),—(CH₂)_(t)(C₅-C₁₀ heteroaryl), —(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and—(CH₂)_(t)(C₅-C₁₀ heterocycloalkyl), wherein t is an integer from 0 to 4and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present,may themselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄ alkoxy, C₁₋₄haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy or hydroxy. Each R″independently represents H or C₁-C₆alkyl.

Examples of representative substituted aryls include the following

In these formulae one of R⁵² and R⁵³ may be hydrogen and at least one ofR⁵² and R⁵³ is each independently selected from alkyl, alkenyl, alkynyl,cycloheteroalkyl, alkanoyl, alkoxy, aryloxy, heteroaryloxy, alkylamino,arylamino, heteroarylamino, NR⁵⁴COR⁵⁵, NR⁵⁴SOR⁵⁵, NR⁵⁴SO₂R⁵⁷, COOalkyl,COOaryl, CONR⁵⁴R⁵⁵, CONR⁵⁴OR⁵⁵, NR⁵⁴R⁵⁵, SO₂NR⁵⁴R⁵⁵, S-alkyl, S-alkyl,SOalkyl, SO₂alkyl, Saryl, SOaryl, SO₂aryl; or R⁵² and R⁵³ may be joinedto form a cyclic ring (saturated or unsaturated) from 5 to 8 atoms,optionally containing one or more heteroatoms selected from the group N,O or S. R⁵⁴, R⁵⁵, and R⁵⁶ are independently hydrogen, alkyl, alkenyl,alkynyl, perfluoroalkyl, cycloalkyl, cycloheteroalkyl, aryl, substitutedaryl, heteroaryl, substituted or hetero alkyl or the like.

‘Hetero’ when used to describe a compound or a group present on acompound means that one or more carbon atoms in the compound or grouphave been replaced by a nitrogen, oxygen, or sulfur heteroatom. Heteromay be applied to any of the hydrocarbyl groups described above such asalkyl, e.g. heteroalkyl, cycloalkyl, e.g. cycloheteroalkyl, aryl, e.g.heteroaryl, cycloalkenyl, cycloheteroalkenyl, and the like having from 1to 5, and especially from 1 to 3 heteroatoms.

‘Heteroaryl’ means an aromatic ring structure, mono-cyclic orpolycyclic, that includes one or more heteroatoms and 5 to 12 ringmembers, more usually 5 to 10 ring members. The heteroaryl group can be,for example, a five membered or six membered monocyclic ring or abicyclic structure formed from fused five and six membered rings or twofused six membered rings or, by way of a further example, two fused fivemembered rings. Each ring may contain up to about four heteroatomstypically selected from nitrogen, sulphur and oxygen. Typically theheteroaryl ring will contain up to 4 heteroatoms, more typically up to 3heteroatoms, more usually up to 2, for example a single heteroatom. Inone embodiment, the heteroaryl ring contains at least one ring nitrogenatom. The nitrogen atoms in the heteroaryl rings can be basic, as in thecase of an imidazole or pyridine, or essentially non-basic as in thecase of an indole or pyrrole nitrogen. In general the number of basicnitrogen atoms present in the heteroaryl group, including any aminogroup substituents of the ring, will be less than five. Examples of fivemembered monocyclic heteroaryl groups include but are not limited topyrrole, furan, thiophene, imidazole, furazan, oxazole, oxadiazole,oxatriazole, isoxazole, thiazole, isothiazole, pyrazole, triazole andtetrazole groups. Examples of six membered monocyclic heteroaryl groupsinclude but are not limited to pyridine, pyrazine, pyridazine,pyrimidine and triazine. Particular examples of bicyclic heteroarylgroups containing a five membered ring fused to another five memberedring include but are not limited to imidazothiazole andimidazoimidazole. Particular examples of bicyclic heteroaryl groupscontaining a six membered ring fused to a five membered ring include butare not limited to benzfuran, benzthiophene, benzimidazole, benzoxazole,isobenzoxazole, benzisoxazole, benzthiazole, benzisothiazole,isobenzofuran, indole, isoindole, isoindolone, indolizine, indoline,isoindoline, purine (e.g., adenine, guanine), indazole,pyrazolopyrimidine, triazolopyrimidine, benzodioxole andpyrazolopyridine groups. Particular examples of bicyclic heteroarylgroups containing two fused six membered rings include but are notlimited to quinoline, isoquinoline, chroman, thiochroman, chromene,isochromene, chroman, isochroman, benzodioxan, quinolizine, benzoxazine,benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline,phthalazine, naphthyridine and pteridine groups. Particular heteroarylgroups are those derived from thiophene, pyrrole, benzothiophene,benzofuran, indole, pyridine, quinoline, imidazole, oxazole andpyrazine.

Examples of representative heteroaryls include the following:

wherein each Y is selected from carbonyl, N, NR⁵⁸, O and S; and R⁵⁸ isindependently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl,heteroaryl and heteroalkyl. The term ‘heteroaryl’ includes‘bicycloheteroaryl’ as defined below.

‘Bicycloheteroaryl’ refers to a monovalent bicycloheteroaromatic groupderived by the removal of one hydrogen atom from a single atom of aparent bicycloheteroaromatic ring system. Typical bicycloheteroarylgroups include, but are not limited to, groups derived from benzofuran,benzimidazole, benzindazole, benzdioxane, chromene, chromane, cinnoline,phthalazine, indole, indoline, indolizine, isobenzofuran, isochromene,isoindole, isoindoline, isoindolone, isoquinoline, benzothiazole,benzoxazole, naphthyridine, benzoxadiazole, pteridine, purine,benzopyran, benzpyrazine, pyridopyrimidine, quinazoline, quinoline,quinolizine, quinoxaline, benzomorphan, tetrahydroisoquinoline andtetrahydroquinoline. Preferably, the bicycloheteroaryl group is between9-11 membered bicycloheteroaryl, with 5-10 membered heteroaryl beingparticularly preferred. Particular bicycloheteroaryl groups are thosederived from benzothiophene, benzofuran, benzothiazole, indole,quinoline, isoquinoline, benzimidazole, benzoxazole and benzdioxane.

As used herein, the term ‘heterocycloalkyl’ refers to a 4-7 membered,stable heterocyclic non-aromatic ring and fused rings containing one ormore heteroatoms independently selected from N, O and S. A fusedheterocyclic ring system may include carbocyclic rings and need onlyinclude one heterocyclic ring. Examples of heterocyclic rings include,but are not limited to, morpholine, piperidine (e.g. 1-piperidinyl,2-piperidinyl, 3-piperidinyl and 4-piperidinyl), pyrrolidine (e.g.1-pyrrolidinyl, 2-pyrrolidinyl and 3-pyrrolidinyl), pyrrolidone, pyran(2H-pyran or 4H-pyran), dihydrothiophene, dihydropyran, dihydrofuran,dihydrothiazole, tetrahydrofuran, tetrahydrothiophene, dioxane,tetrahydropyran (e.g. 4-tetrahydro pyranyl), imidazoline,imidazolidinone, oxazoline, thiazoline, 2-pyrazoline, pyrazolidine,piperazine, and N-alkyl piperazines such as N-methyl piperazine. Furtherexamples include thiomorpholine and its S-oxide and S,S-dioxide(particularly thiomorpholine). Still further examples include azetidine,piperidone, piperazone, and N-alkyl piperidines such as N-methylpiperidine. Particular examples of heterocycloalkyl groups are shown inthe following illustrative examples:

wherein each X is selected from CR⁵⁸, CR⁵⁸ ₂, NR⁵⁸, O and S; and each Yis selected from NR⁵⁸, O and S; and R⁵⁸ is independently hydrogen,alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, heteroalkyl orthe like. These heterocycloalkyl rings may be optionally substitutedwith one or more groups selected from the group consisting of acyl,acylamino, acyloxy, alkoxy, substituted alkoxy, alkoxycarbonyl,alkoxycarbonylamino, amino, substituted amino, aminocarbonyl,aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, carboxyl,cyano, cycloalkyl, substituted cycloalkyl, halogen, hydroxyl, keto,nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioketo, thiol,alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— and aryl-S(O)₂—. Substitutinggroups include carbonyl or thiocarbonyl which provide, for example,lactam and urea derivatives. The term ‘heterocycloalkyl’ includes‘heterocycloalkenyl’ as defined below.

Examples of representative heterocycloalkenyls include the following:

wherein each X is selected from CR⁵⁸, CR⁵⁸ ₂, NR⁵⁸, O and S; and each Yis selected from carbonyl, N, NR⁵⁸, O and S; and R⁵⁸ is independentlyhydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl,heteroalkyl or the like.

Examples of representative aryl having hetero atoms containingsubstitution include the following:

wherein each X is selected from CR⁵⁸ ₂, NR⁵⁸, O and S; and each Y isselected from carbonyl, NR⁵⁸, O and S; and R⁵⁸ is independentlyhydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl,heteroalkyl or the like.

‘Hetero substituent’ refers to a halo, O, S or N atom-containingfunctionality that may be present as an ‘R’ in a ‘R’C group present assubstituents directly on a variable ring atom of the compounds of thisinvention or may be present as a substituent in the “substituted” aryland aliphatic groups present in the compounds. Examples of heterosubstituents include:

-   -   -halo,    -   —NO₂, —NH₂, —NHR⁵⁹, —N(R⁵⁹)₂,    -   —NRCOR, —NR⁵⁹SOR⁵⁹, —NR⁵⁹SO₂R⁵⁹, OH, CN,    -   —CO₂H,    -   —R⁵⁹—OH, —COOR⁵⁹,    -   —CON(R⁵⁹)₂, —CONROR⁵⁹,    -   —SO₃H, —SO₂N(R⁵⁹)₂,    -   —S(O)R⁵⁹, —S(O)₂R⁵⁹

wherein each R⁵⁹ is independently an aryl or aliphatic, optionally withsubstitution. Among hetero substituents containing R⁵⁹ groups,preference is given to those materials having aryl and alkyl R⁵⁹ groupsas defined herein. Preferred hetero substituents are those listed above.

‘Hydrogen bond donor’ group refers to a group containing O—H, N—Hfunctionality. Examples of “hydrogen bond donor” groups include —OH,—NH₂, and —NH—R^(59a), wherein R^(59a) is alkyl, cycloalkyl, aryl orheteroaryl.

‘Dihydroxyphosphoryl’ refers to the radical —PO(OH)₂.

‘Substituted dihydroxyphosphoryl’ refers to a dihydroxyphosphoryl groupsubstituted with one or more of those groups recited in the definitionof ‘substituted’ herein, and particularly refers to adihydroxyphosphoryl radical wherein one or both of the hydroxyl groupsare substituted.

‘Aminohydroxyphosphoryl’ refers to the radical —PO(OH)NH₂.

‘Substituted aminohydroxyphosphoryl’ refers to an aminohydroxyphosphorylgroup substituted with one or more of those groups recited in thedefinition of ‘substituted’ herein, and particularly refers to anaminohydroxyphosphoryl wherein the amino group is substituted with oneor two substituents. In certain embodiments, the hydroxyl group can alsobe substituted.

‘Thioalkoxy’ refers to the group —SR⁶⁰ where R⁶⁰ is alkyl.

‘Substituted thioalkoxy’ refers to a thioalkoxy group substituted withone or more of those groups recited in the definition of ‘substituted’herein, and particularly refers to a thioalkoxy group having 1 or moresubstituents, for instance from 1 to 5 substituents, and particularlyfrom 1 to 3 substituents.

‘Sulfanyl’ refers to the radical HS—. ‘Substituted sulfanyl’ refers to asulfanyl group substituted with one or more of those groups recited inthe definition of ‘substituted’ herein. Exemplary ‘substituted sulfanyl’groups are —S—C₁-C₈ alkyl, —S—(CH₂)_(t)(C₆-C₁₀ aryl),—S—(CH₂)_(t)(C₅-C₁₀ heteroaryl), —S—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and—S—(CH₂)_(t)(C₅-C₁₀ heterocycloalkyl), wherein t is an integer from 0 to4 and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groupspresent, may themselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄alkoxy, C₁₋₄ haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy orhydroxy.

‘Sulfonyl’ refers to the divalent radical —S(O₂)—. ‘Substitutedsulfonyl’ refers to a radical such as —S(O₂)—R⁶¹, wherein R⁶¹ is anysubstituent described herein. Exemplary ‘substituted sulfonyl’ groupsare —SO₂—C₁-C₈ alkyl, —SO₂—(CH₂)_(t)(C₆-C₁₀ aryl), —SO₂—(CH₂)_(t)(C₅-C₁₀heteroaryl), —SO₂—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and—SO₂—(CH₂)_(t)(C₅-C₁₀ heterocycloalkyl), wherein t is an integer from 0to 4 and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groupspresent, may themselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄alkoxy, C₁-₄ haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy orhydroxy.

“Aminosulfonyl” or “Sulfonamide” refers to the radical —S(O₂)NH₂, and“substituted aminosulfonyl” or “substituted sulfonamide” refers to aradical such as —S(O₂)NR⁶² ₂ wherein each R⁶² is independently anysubstituent described herein.

Exemplary “substituted aminosulfonyl” or “substituted sulfonamide”groups are —S(O₂)N(R⁶²)—C₁-C₈ alkyl, —S(O₂)N(R⁶²)—(CH₂)_(t)(C₆-C₁₀aryl), —S(O₂)N(R⁶²)—(CH₂)_(t)(C₅-C₁₀ heteroaryl),—S(O₂)N(R⁶²)—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and—S(O₂)N(R⁶²)—(CH₂)_(t)(C₅-C₁₀ heterocycloalkyl), wherein t is an integerfrom 0 to 4; each R⁶² independently represents H or C₁-C₆ alkyl; and anyaryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, maythemselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄ alkoxy, C₁₋₄haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy or hydroxy. EachR^(B′) independently represents H or C₁-C₆ alkyl.

‘Sulfone’ refers to the group —SO₂R⁶³. In particular embodiments, R⁶³ isselected from H, lower alkyl, alkyl, aryl and heteroaryl.

“Sulfo” or “sulfonic acid” refers to a radical such as —SO₃H.

“Substituted sulfo” or “sulfonic acid ester” refers to a radical such as—SO₃R^(61b) wherein R^(61b) is substituted or unsubstituted alkyl orsubstituted or unsubstituted aryl. Exemplary “Substituted Sulfo” or“sulfonic acid ester” groups are SO₃—C₁-C₈ alkyl, SO₃—(CH₂)_(t)(C₆-C₁₀aryl), SO₃—(CH₂)_(t)(C₅-C₁₀ heteroaryl), SO₃—(CH₂)_(t)(C₃-C₁₀cycloalkyl), and SO₃—(CH₂)_(t)(C₅-C₁₀ heterocycloalkyl), wherein t is aninteger from 0 to 4 and any aryl, heteroaryl, cycloalkyl orheterocycloalkyl groups present, may themselves be substituted by C₁-C₄alkyl, halo, C₁-C₄ alkoxy, C₁₋₄ haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄haloalkoxy or hydroxy. “Sulfinyl” refers to the divalent radical —S(O)—.“Substituted sulfinyl” refers to a radical such as —SOR^(61a), whereinR^(61a) is any substituent described herein. Exemplary “substitutedsulfinyl” groups are SO—C₁-C₈ alkyl, SO—(CH₂)_(t)(C₆-C₁₀ aryl),SO—(CH₂)_(t)(C₅-C₁₀ heteroaryl). SO—(CH₂)_(t)(C₅-C₁₀ cycloalkyl), andSO—(CH₂)_(t)(C₅-C₁₀ heterocycloalkyl), wherein t is an integer from 0 to4 and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groupspresent, may themselves be substituted by C₁-C₄ alkyl, halo, C₁-C₄alkoxy, C₁₋₄ haloalkyl, C₁-C₄ hydroxyalkyl, or C₁-C₄ haloalkoxy orhydroxy.

‘Thioaryloxy’ refers to the group —SR⁶⁴ where R⁶⁴ is aryl.

‘Thioketo’ refers to the group ═S.

‘Thiol’ refers to the group —SH.

One having ordinary skill in the art of organic synthesis will recognizethat the maximum number of heteroatoms in a stable, chemically feasibleheterocyclic ring, whether it is aromatic or non aromatic, is determinedby the size of the ring, the degree of unsaturation and the valence ofthe heteroatoms. In general, a heterocyclic ring may have one to fourheteroatoms so long as the heteroaromatic ring is chemically feasibleand stable.

‘Pharmaceutically acceptable’ means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

‘Pharmaceutically acceptable salt’ refers to a salt of a compound of theinvention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. In particular,such salts are non-toxic may be inorganic or organic acid addition saltsand base addition salts. Specifically, such salts include: (1) acidaddition salts, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or formed with organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike. Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe compound contains a basic functionality, salts of non toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like. The term“pharmaceutically acceptable cation” refers to an acceptable cationiccounter-ion of an acidic functional group. Such cations are exemplifiedby sodium, potassium, calcium, magnesium, ammonium, tetraalkylammoniumcations, and the like.

‘Pharmaceutically acceptable vehicle’ refers to a diluent, adjuvant,excipient or carrier with which a compound of the invention isadministered.

‘Preventing’ or ‘prevention’ refers to a reduction in risk of acquiringa disease or disorder (i.e., causing at least one of the clinicalsymptoms of the disease not to develop in a subject that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease). This term encompasses the term ‘prophylaxis’,which means a measure taken for the prevention of a disease.

‘Prodrugs’ refers to compounds, including derivatives of the compoundsof the invention, which have cleavable groups and become by solvolysisor under physiological conditions the compounds of the invention whichare pharmaceutically active in vivo. Such examples include, but are notlimited to, choline ester derivatives and the like, N-alkylmorpholineesters and the like.

‘Solvate’ refers to forms of the compound that are associated with asolvent, usually by a solvolysis reaction. This physical associationincludes hydrogen bonding. Conventional solvents include water, ethanol,acetic acid and the like. The compounds of the invention may be preparede.g. in crystalline form and may be solvated or hydrated. Suitablesolvates include pharmaceutically acceptable solvates, such as hydrates,and further include both stoichiometric solvates and non-stoichiometricsolvates. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. ‘Solvate’ encompasses bothsolution-phase and isolable solvates. Representative solvates includehydrates, ethanolates and methanolates.

‘Subject’ includes humans. The terms ‘human’, ‘patient’ and ‘subject’are used interchangeably herein.

‘Therapeutically effective amount’ means the amount of a compound that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” can vary depending on the compound, the disease and itsseverity, and the age, weight, etc., of the subject to be treated. Inparticular, with regard to treating an disease involving cartilage orjoint degradation and/or inflammation, the terms “therapeuticallyeffective amount” or “effective MAPKAPK5-inhibiting amount” is intendedto mean that effective amount of an compound of the present inventionthat will bring about a biologically meaningful decrease in MAPKAPK5expression or activity in the subject's disease affected tissues, suchthat cartilage or joint degradation and/or inflammation is meaningfullyreduced. A compound having MAPKAPK5-inhibiting properties or a“MAPKAPK5-inhibiting compound” means a compound of the present inventionthat provided to a cell in effective amounts is able to cause abiologically meaningful decrease in MAPKAPK5 expression or activity insuch cells.

‘Treating’ or ‘treatment’ of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e., arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment “treating” or “treatment”refers to ameliorating at least one physical parameter, which may not bediscernible by the subject. In yet another embodiment, “treating” or“treatment” refers to modulating the disease or disorder, eitherphysically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In yet another embodiment, ‘treating’ or ‘treatment’ refers to delayingthe onset of the disease or disorder.

‘Compounds of the present invention’, and equivalent expressions, aremeant to embrace compounds of the Formula (e) as hereinbefore described,which expression includes the prodrugs, the pharmaceutically acceptablesalts, and the solvates, e.g., hydrates, where the context so permits.Similarly, reference to intermediates, whether or not they themselvesare claimed, is meant to embrace their salts, and solvates, where thecontext so permits.

Other derivatives of the compounds of this invention have activity inboth their acid and acid derivative forms, but in the acid sensitiveform often offers advantages of solubility, tissue compatibility, ordelayed release in the mammalian organism (see, Bundgard, H., Design ofProdrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs includeacid derivatives well know to practitioners of the art, such as, forexample, esters prepared by reaction of the parent acid with a suitablealcohol, or amides prepared by reaction of the parent acid compound witha substituted or unsubstituted amine, or acid anhydrides, or mixedanhydrides. Simple aliphatic or aromatic esters, amides and anhydridesderived from acidic groups pendant on the compounds of this inventionare particularly useful prodrugs. In some cases it is desirable toprepare double ester type prodrugs such as (acyloxy)alkyl esters or((alkoxycarbonyl)oxy)alkylesters. Particular such prodrugs are the C₁ toC₈ alkyl, C₂-C₈ alkenyl, aryl, C₇-C₁₂ substituted aryl, andC₁-C₁₂arylalkyl esters of the compounds of the invention.

As used herein, the term ‘isotopic variant’ refers to a compound thatcontains unnatural proportions of isotopes at one or more of the atomsthat constitute such compound. For example, an ‘isotopic variant’ of acompound can contain one or more non-radioactive isotopes, such as forexample, deuterium (²H or D), carbon-13 (¹³C), nitrogen-15 (¹⁵N), or thelike. It will be understood that, in a compound where such isotopicsubstitution is made, the following atoms, where present, may vary, sothat for example, any hydrogen may be ²H/D, any carbon may be ¹³C, orany nitrogen may be ¹⁵N, and that the presence and placement of suchatoms may be determined within skill of the art. Likewise, the inventionmay include the preparation of isotopic variants with radioisotopes, inthe instance for example, where the resulting compounds may be used fordrug and/or substrate tissue distribution studies. The radioactiveisotopes tritium, i.e. ³H, and carbon-14, i.e. ¹⁴C, are particularlyuseful for this purpose in view of their ease of incorporation and readymeans of detection. Further, compounds may be prepared that aresubstituted with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, and would be useful in Positron Emission Topography (PET) studiesfor examining substrate receptor occupancy.

All isotopic variants of the compounds provided herein, radioactive ornot, are intended to be encompassed within the scope of the invention.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed ‘isomers’. Isomersthat differ in the arrangement of their atoms in space are termed‘stereoisomers’.

Stereoisomers that are not mirror images of one another are termed‘diastereomers’ and those that are non-superimposable mirror images ofeach other are termed ‘enantiomers’. When a compound has an asymmetriccenter, for example, it is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a ‘racemic mixture’.

‘Tautomers’ refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of π electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the aci- and nitro-forms of phenylnitromethane, that arelikewise formed by treatment with acid or base.

Tautomeric forms may be relevant to the attainment of the optimalchemical reactivity and biological activity of a compound of interest.

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof.

Unless indicated otherwise, the description or naming of a particularcompound in the specification and claims is intended to include bothindividual enantiomers and mixtures, racemic or otherwise, thereof. Themethods for the determination of stereochemistry and the separation ofstereoisomers are well-known in the art.

The Compounds

The present invention is based on the discovery of that MAPKAPK5functions in the pathway that results in the expression of MMP1, andthat inhibitors of MAPKAPK5 activity, such as the compounds of thepresent invention, are useful for the treatment of diseases involvingthe abnormally high expression of MMP activity.

The compounds of the present invention may be described generally asfused pyrazines, in particular imidazo[1,2-a]pyrazines and[1.2.4]triazolo[1,5-a]pyrazine substituted in the 5-position by an aryland heteroaryl group, and an in the 8-position by a heteroarylaminogroup.

More particularly, the present invention relates to compounds accordingto formula (I):

wherein

Z is CH or N; R¹ is H, or substituted or unsubstituted C₁-C₆ alkyl; R²is H, C₁-C₆ alkyl, C₃-C₇ cycloalkyl or C₃-C₇ cycloalkyl-C₁-C₆ alkyl,optionally substituted with one or more of F and Cl; R⁸ is substitutedor unsubstituted heteroaryl; R⁹ is selected from substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl; or apharmaceutically acceptable salt, solvate or prodrug thereof; andstereoisomers, isotopic variants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, R¹ is H.

In one embodiment, with respect to compounds of formula I, R² is H.

In one embodiment, with respect to compounds of formula I, R⁸ issubstituted or unsubstituted 5-membered heteroaryl.

In another embodiment, with respect to compounds of formula I, R⁸ isselected from substituted or unsubstituted pyrrolyl, furanyl, thienyl,pyrazolyl, oxazolyl, imidazolyl, thiazolyl, 1,3,4-thiadiazolyl, and1,2,4-thiadiazolyl.

In another embodiment, with respect to compounds of formula I, R⁸ isselected from substituted pyrrolyl, furanyl, thienyl, pyrazolyl,oxazolyl, imidazolyl, thiazolyl, 1,3,4-thiadiazolyl, and1,2,4-thiadiazolyl and the substitution is selected from C₁-C₆ alkyl,substituted C₁-C₆ alkyl, carbamoyl, C₁-C₆ hydroxyalkyl, C₁-C₆ haloalkyl,C₁-C₆ alkoxy, cyano, amino, sulfo, aryl, C₃-C₇ cycloalkyl, aralkyl,heterocycloalkyl and heteroaryl.

In one embodiment, with respect to compounds of formula I, R⁸ isselected from substituted pyrrolyl, furanyl, thienyl, pyrazolyl,oxazolyl, imidazolyl, thiazolyl, 1,3,4-thiadiazolyl, and1,2,4-thiadiazolyl and the substitution is selected from is Me, i-Pr,cyclopropyl, CH₂OH, CONH₂, CONMe₂, NMe₂, NEt₂, SO₂Me, or SO₂Et.

In one embodiment, with respect to compounds of formula I, R⁸ is

wherein R⁸ is unsubstituted or substituted with C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, carbamoyl, C₁-C₆ hydroxyalkyl, C₁-C₆ haloalkyl, C₁-C₆alkoxy, cyano, amino, sulfo, aryl, 3-7-membered heterocycloalkyl,aralkyl, heterocycloalkyl and heteroaryl; and wherein R^(8c) isindependently selected from hydrogen, and substituted or unsubstitutedC₁-C₆ alkyl.

In one embodiment, with respect to compounds of formula I, R⁸ is

wherein Cy′ is C₃-C₇ cycloalkyl, heterocycloalkyl, aryl or heteroarylring; and R^(8c) is independently selected from hydrogen, andunsubstituted or substituted C₁-C₆ alkyl.

In one embodiment, with respect to compounds of formula I, R⁸ is

wherein R^(8c) is independently selected from hydrogen, andunsubstituted or substituted C₁-C₆ alkyl.

In one embodiment, with respect to compounds of formula I, R⁸ isselected from substituted pyrrolyl, furanyl, thienyl, pyrazolyl,oxazolyl, imidazolyl, thiazolyl, 1,3,4-thiadiazolyl, and1,2,4-thiadiazolyl and the substitution is -L-R^(8d);

wherein

-   -   L is selected from bond, alkylene, heteroalkylene, —O—,        —N(R^(8c))—, —CO—, —CO₂—, —SO—, —SO₂—, —CON(R^(8e))—,        —SO₂N(R^(8e))—, —N(R^(8e))CO—, —N(e)SO₂—, —N(R^(8e))CI        N(R^(8e))—, —N(R^(8e))SO₂N(R^(8e))—; and    -   R^(8d) is selected from substituted or unsubstituted C₁-C₆        alkyl, substituted or unsubstituted C₃-C₇ cycloalkyl,        substituted or unsubstituted aryl, substituted or unsubstituted        3-7-membered heterocycloalkyl, substituted or unsubstituted        heteroaryl, substituted or unsubstituted amino, substituted or        unsubstituted aralkyl, substituted or unsubstituted        heteroarylalkyl and substituted or unsubstituted aminoalkyl; and    -   R^(8e) is selected from H, substituted or unsubstituted C₁-C₆        alkyl and substituted or unsubstituted C₃-C₇ cycloalkyl.

In one embodiment, with respect to compounds of formula I, R^(8c) is-L-R^(8d)

In one embodiment, with respect to compounds of formula I, R⁸ is

-   -   wherein L, and R^(8d) are as described in the preceding        paragraph; and R^(8c) is independently selected from hydrogen,        and substituted or unsubstituted C₁-C₆ alkyl.

In one embodiment, with respect to compounds of formula I, R⁸ is asdescribed above; L is —CON(R^(8e))— or SO₂N(R^(8e))—;

-   -   R^(8d) is selected from substituted or unsubstituted C₁-C₆        alkyl, substituted or unsubstituted C₃-C₇ cycloalkyl,        substituted or unsubstituted aryl, substituted or unsubstituted        3-7-membered heterocycloalkyl, substituted or unsubstituted        heteroaryl, substituted or unsubstituted aralkyl, substituted or        unsubstituted C₁-C₆ heteroarylalkyl and substituted or        unsubstituted C₁-C₆ aminoalkyl; and    -   R^(8e) is selected from H, substituted or unsubstituted C₁-C₆        alkyl.

In one embodiment, with respect to compounds of formula I, R⁸ is L is—CONH— or SO₂NH—; and

-   -   R^(8d) is selected from H, C₁-C₆ alkylaminoethyl, diC₁-C₆        alkylaminoethyl, C₃-C₇ cycloalkyl, 3-7-membered        heterocycloalkyl, arylalkyl, and heteroarylalkyl.

In one embodiment, with respect to compounds of formula I, R⁸ is L is—CONH— or SO₂NH—; and R^(8d) is selected from H, methylaminoethyl,ethylaminoethyl, dimethylaminoethyl, diethylaminoethyl, substituted orunsubstituted pyrrolidinyl, benzyl and pyridylmethyl.

In one embodiment, with respect to compounds of formula I, R⁸ is L isbond, —CO—, SO₂, —(CH₂)_(m1)—, —O(CH₂)_(m1)—, —NH(CH₂)_(m1)—,—CON(H)(CH₂)_(m1)—, or —SO₂NH(CH₂)_(m1)—; the subscript m1 is selectedfrom 1-4; and R^(8d) is

-   -   wherein the ring P is substituted or unsubstituted 3-7-membered        heterocycloalkyl.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula IIa, IIb, IIe, IId, IIe or IIf:

and wherein L, R^(8c), and the ring P are as described above; and R⁹ isindependently selected from substituted or unsubstituted aryl andheteroaryl; or a pharmaceutically acceptable salt, solvate or prodrugthereof; and stereoisomers, isotopic variants and tautomers thereof.

In one embodiment, with respect to compounds of formulae IIa-IIf, L is abond.

In one embodiment, with respect to compounds of formulae L is —CO—.

In one embodiment, with respect to compounds of formulae IIa-IIf, L is—SO₂—.

In one embodiment, with respect to compounds of formulae IIa-IIf, L is—CON(H)—CH₂—CH₂—, or —SO₂NH—CH₂—CH₂—.

In one embodiment, with respect to compounds of formulae IIa-IIf, L is—OCH₂—CH₂— or —NHCH₂—CH₂—.

In one embodiment, with respect to compounds of formulae IIa-IIf thering P is substituted or unsubstituted piperidine, morpholine orpiperazine.

In one embodiment, with respect to compounds of formulae IIa-IIf, thering P is substituted or unsubstituted:

-   -   and R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl        or substituted or unsubstituted C₃-C₇ cycloalkyl.

In one embodiment, with respect to compounds of formulae the ring P isas described above and R^(8b) is H, Me, Et, Pr, i-Pr, t-Bu, i-Bu,CH₂CONH₂, or cyclopropylmethyl.

In one embodiment, with respect to compounds of formulae IIa-IIf, thering P is as described above and R^(8b) is substituted or unsubstitutedC₃-C₇ cycloalkyl. In yet another embodiment, R^(8b) is cyclohexyl,cyclopentyl. In further embodiments, R^(8b) is cyclopropyl.

In further embodiments, with respect to compounds of formula I, thecompound is according to formula IIIa, IIIb, IIIc, IIId, IIIe, IIIf,IIIg, IIIh, IIIi, Illj, IIIk, IIIl, or IIIm:

and wherein L is a bond, CO, or substituted or unsubstituted C₁-C₄alkylene; R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; and R⁹ isindependently selected from substituted or unsubstituted aryl andheteroaryl; or a pharmaceutically acceptable salt, solvate or prodrugthereof; and stereoisomers, isotopic variants and tautomers thereof.

In further embodiments, with respect to compounds of formula I, thecompound is according to formulae IIIn, IIIo, IIIp, IIIq, IIIs, IIIt,IIIu, IIIv, IIIw, IIIx, or IIIy:

and wherein L is a bond, CO, or substituted or unsubstituted C₁-C₄alkylene; R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; and R⁹ isindependently selected from substituted or unsubstituted aryl andheteroaryl; or a pharmaceutically acceptable salt, solvate or prodrugthereof; and stereoisomers, isotopic variants and tautomers thereof.

In further embodiments, with respect to compounds of formula I, thecompound is according to formula IIIz, IIIaa, IIIab, IIIac, IIIad, orIIIae:

and wherein L is a bond, CO, or substituted or unsubstituted C₁-C₄alkylene; R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; and R⁹ isindependently selected from substituted or unsubstituted aryl andheteroaryl; or a pharmaceutically acceptable salt, solvate or prodrugthereof; and stereoisomers, isotopic variants and tautomers thereof.

In one embodiment, with respect to compounds of formulae IIIa-ae, L is abond.

In one embodiment, with respect to compounds of formulae IIIa-ae, L is—CH₂—.

In one embodiment, with respect to compounds of formulae IIIa-ae, L is—CO—.

In one embodiment, with respect to compounds of formulae IIIa-ae, L is—SO₂—.

In one embodiment, with respect to compounds of formulae IIIa-ae, L is—CON(H)—CH₂—CH₂—, or —SO₂NH—CH₂—CH₂—.

In one embodiment, with respect to compounds of formulae IIIa-ae, L is—OCH₂—CH₂— or —NHCH₂—CH₂—.

In one embodiment, with respect to compounds of formulae IIIa-ae, R^(8b)is H. In another embodiment, R^(8b) is substituted or unsubstitutedalkyl. In yet another embodiment, R^(8b) is Me, Et, Pr, i-Pr, t-Bu,i-Bu, CH₂CONH₂, or cyclopropylmethyl.

In one embodiment, R^(8b) is substituted or unsubstituted cycloalkyl. Inyet another embodiment, R^(8b) is cyclohexyl, cyclopentyl. In furtherembodiments, R^(8b) is cyclopropyl.

In one embodiment, with respect to compounds of formula I, R⁸ is fusedheteroaryl according to compounds of formulae IIIaf, IIIag, or IIIah

and wherein R^(8c) is hydrogen, or substituted or unsubstitutedC₁-C₆alkyl.

In one embodiment, with respect to compounds of formulae I-IIIr, R⁹ isselected from substituted or unsubstituted phenyl, pyridyl, indolyl,isoindolyl, pyrrolyl, furanyl, thienyl, pyrazolyl, oxazolyl, andthiazolyl.

In one embodiment, with respect to compounds of formulae I-IIIah, R⁹ is

-   -   and each of A¹, A² and A³ is independently selected from S, O,        N, NR^(9a), and CR^(9a); each of R^(9a) is independently H or        substituted or unsubstituted C₁-C₆ alkyl; and R^(9b) is CONH₂,        CONHMe, or CN.

In further embodiment, with respect to compounds of formulae I-IIIr, R⁹is

In further embodiment, with respect to compounds of formulae I-IIIr, R⁹is

In further embodiment, with respect to compounds of formulae I-IIIr, R⁹is

-   -   wherein the subscript m is selected from 0-4 and each R^(9d) is        independently substituted or unsubstituted C₁-C₆ alkyl or halo.

In a further embodiment, with respect to compounds of formulae I-IIIr,R⁹ is

-   -   wherein the subscript m is selected from 0-3 and each R^(9d) is        independently substituted or unsubstituted alkyl or halo.

In a further embodiment, with respect to compounds of formulae I-IIIr,R⁹ is as described above; m is 1 or 2 and each R^(9d) is independentlyMe, Cl or F.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVi,IVj, IVk or IVl:

and wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers, isotopicvariants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula IVm, IVn, IVo, IVp, IVq, IVr, IVs, IVt, IVu,IVv, IVw or IVx:

and wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers, isotopicvariants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula Va, Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk orVl:

and wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; and R^(9e) ishydrogen, C₁-C₆ alkyl, amino, amido, or CN; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers, isotopicvariants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula Vm, Vn, Vo, Vp, Vq, Vr, Vs, Vt, Vu, Vv, Vw orVx:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; and R^(9e) is hydrogen,C₁-C₆ alkyl, amino, amido, or CN; or a pharmaceutically acceptable salt,solvate or prodrug thereof; and stereoisomers, isotopic variants andtautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula VIa, VIb, VIc, VId, VIe, VIf, VIg, VIh, VIi,VIj, VIk or VIl:

and wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers, isotopicvariants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula VIm, VIn, VIo, VIp, VIq, VIr, VIs, VIt, VIu,VIv, VIw or VIx:

and wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers, isotopicvariants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula VIIa, VIIb, VIIc, VIId, VIIe, VIIf, VIIg, VIIh,VIIi, VIIj, VIIk or VIIl:

and wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers, isotopicvariants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula VIIm, VIIn, VIIo, VIIp, VIIq, VIIr, VIIs, VIIt,VIIu, VIIv, VIIw or VIIx:

and wherein R^(8b) is hydrogen; substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₁-C₆ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers, isotopicvariants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula VIIIa, VIIIb, VIIIc, VIIId, VIIIe, VIII, VIIIg,VIIIh, VIIIi, VIIIj, VIIIk or VIIIl:

-   -   and wherein R^(8b) is hydrogen, substituted or unsubstituted        C₁-C₆ alkyl or substituted or unsubstituted C₃-C₇ cycloalkyl;        and R^(9e) is hydrogen, C₁-C₆ alkyl, amino, amido, or CN; or a        pharmaceutically acceptable salt, solvate or prodrug thereof;        and stereoisomers, isotopic variants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula VIIIm, VIIIn, VIIIo, VIIIp, VIIIq, VIIIr, VIIIs,VIIIt, VIIIu, VIIIv, VIIIw or VIIIx:

and wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; and R^(9e) ishydrogen, C₁-C₆ alkyl, amino, amido, or CN; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers, isotopicvariants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula IXa, IXb, IXc, IXd, IXe, IXf, IXg, IXh, IXi,IXj, IXk or IXl:

and wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers, isotopicvariants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula IXm, IXn, IXo, IXp, IXq, IXr, IXs, IXt, IXu,IXv, IXw or IXx:

and wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkylor substituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers, isotopicvariants and tautomers thereof.

In one embodiment, with respect to compounds of formulae IVa-IXl, R^(8b)is H.

In one embodiment, with respect to compounds of formulae IVa-IXl, R^(8b)is cycloalkyl.

In one embodiment, with respect to compounds of formulae IVa-IXl, R^(8b)is cyclopropyl.

In one embodiment, with respect to compounds of formulae IVa-IXl, R^(8b)is substituted or unsubstituted C₁-C₆ alkyl.

In one embodiment, with respect to compounds of formulae IVa-IXl, R^(8b)is Me, Et, Pr, i-Pr, t-Bu, i-Bu, CF₃, CH₂CF₃, or cyclopropylmethyl.

In one embodiment, with respect to compounds of formulae Va-I andVIIIa-l, R^(9e) is H. In another embodiment, R^(9e) is Me. In yetanother embodiment, R^(9e) is CN or CONH₂.

In one embodiment, with respect to formula I, the compound is accordingto formula Xa, Xb or Xc:

and wherein R^(8c) is hydrogen, or substituted or unsubstituted C₁-C₆alkyl.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula Xd, Xe or Xf:

and wherein R^(8c) is hydrogen, or substituted or unsubstituted C₁-C₆alkyl; and R^(9e) is hydrogen, C₁-C₆ alkyl, amino, amido, or CN; or apharmaceutically acceptable salt, solvate or prodrug thereof; andstereoisomers, isotopic variants and tautomers thereof.

In one embodiment, with respect to compounds of formula I, the compoundis according to formula Xg, Xh or Xi:

wherein R^(8c) is hydrogen, or substituted or unsubstituted C₁-C₆ alkyl;and R^(9e) is hydrogen, C₁-C₆ alkyl, amino, amido, or CN; or apharmaceutically acceptable salt, solvate or prodrug thereof; andstereoisomers, isotopic variants and tautomers thereof.

In one embodiment, with respect to compounds of formulae I-Xi, Z is CH.

In one embodiment, with respect to compounds of formulae I-Xi, Z is N.

In certain aspects, the present invention provides prodrugs andderivatives of the compounds according to the formulae above. Prodrugsare derivatives of the compounds of the invention, which havemetabolically cleavable groups and become by solvolysis or underphysiological conditions the compounds of the invention, which arepharmaceutically active, in vivo. A prodrug may be inactive whenadministered to a subject but is converted in vivo to an active compoundof the invention. “Pharmaceutically acceptable prodrugs” as used hereinrefers to those prodrugs of the compounds useful in the presentinvention, which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of patients with unduetoxicity, irritation, allergic response commensurate with a reasonablebenefit/risk ratio, and effective for their intended use of thecompounds of the invention. The term “prodrug” means a compound that istransformed in vivo to yield an effective compound useful in the presentinvention or a pharmaceutically acceptable salt, hydrate or solvatethereof. The transformation may occur by various mechanisms, such asthrough hydrolysis in blood. The compounds bearing metabolicallycleavable groups have the advantage that they may exhibit improvedbioavailability as a result of enhanced solubility and/or rate ofabsorption conferred upon the parent compound by virtue of the presenceof the metabolically cleavable group, thus, such compounds act aspro-drugs. A thorough discussion is provided in Design of Prodrugs, H.Bundgard, ed., Elsevier (1985); Methods in Enzymology; K. Widder et al,Ed., Academic Press, 42, 309-396 (1985); A Textbook of Drug Design andDevelopment, Krogsgaard-Larsen and H. Bundgard, ed., Chapter 5; “Designand Applications of Prodrugs” 113-191 (1991); Advanced Drug DeliveryReviews, H. Bundgard, 8, 1-38, (1992); J. Pharm. Sci., 77,285 (1988);Chem. Pharm. Bull., N. Nakeya et al, 32, 692 (1984); Pro-drugs as NovelDelivery Systems, T. Higuchi and V. Stella, 14 A.C.S. Symposium Series,and Bioreversible Carriers in Drug Design, E. B. Roche, ed., AmericanPharmaceutical Association and Pergamon Press, 1987, which areincorporated herein by reference. Such examples include, but are notlimited to, choline ester derivatives and the like, N-alkylmorpholineesters and the like.

Other derivatives of the compounds of this invention have activity inboth their acid and acid derivative forms, but the acid sensitive formoften offers advantages of solubility, tissue compatibility, or delayedrelease in the mammalian organism (see, Bundgard, H., Design ofProdrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs includeacid derivatives well know to practitioners of the art, such as, forexample, esters prepared by reaction of the parent acid with a suitablealcohol, or amides prepared by reaction of the parent acid compound witha substituted or unsubstituted amine, or acid anhydrides, or mixedanhydrides. Simple aliphatic or aromatic esters, amides and anhydridesderived from acidic groups pendant on the compounds of this inventionare preferred prodrugs. In some cases it is desirable to prepare doubleester type prodrugs such as (acyloxy)alkyl esters or((alkoxycarbonyl)oxy)alkylesters. Preferred are the C₁ to C₈ alkyl,C₂-C₈ alkenyl, aryl, C₇-C₁₂ substituted aryl, and C₇-C₁₂ arylalkylesters of the compounds of the invention.

Pharmaceutical Compositions

When employed as pharmaceuticals, the compounds of this invention aretypically administered in the form of a pharmaceutical composition. Suchcompositions can be prepared in a manner well known in thepharmaceutical art and comprise at least one active compound.

Generally, the compounds of this invention are administered in apharmaceutically effective amount. The amount of the compound actuallyadministered will typically be determined by a physician, in the lightof the relevant circumstances, including the condition to be treated,the chosen route of administration, the actual compound-administered,the age, weight, and response of the individual patient, the severity ofthe patient's symptoms, and the like.

The pharmaceutical compositions of this invention can be administered bya variety of routes including oral, rectal, transdermal, subcutaneous,intravenous, intra-articular, intramuscular, and intranasal. Dependingon the intended route of delivery, the compounds of this invention arepreferably formulated as either injectable or oral compositions or assalves, as lotions or as patches all for transdermal administration.

The compositions for oral administration can take the form of bulkliquid solutions or suspensions, or bulk powders. More commonly,however, the compositions are presented in unit dosage forms tofacilitate accurate dosing. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient. Typical unitdosage forms include prefilled, premeasured ampules or syringes of theliquid compositions or pills, tablets, capsules or the like in the caseof solid compositions. In such compositions, the fuzed pyrazine compoundis usually a minor component (from about 0.1 to about 50% by weight orpreferably from about 1 to about 40% by weight) with the remainder beingvarious vehicles or carriers and processing aids helpful for forming thedesired dosing form.

Liquid forms suitable for oral administration may include a suitableaqueous or nonaqueous vehicle with buffers, suspending and dispensingagents, colorants, flavors and the like. Solid forms may include, forexample, any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based upon injectable sterilesaline or phosphate-buffered saline or other injectable carriers knownin the art. As before, the active compound in such compositions istypically a minor component, often being from about 0.05 to 10% byweight with the remainder being the injectable carrier and the like.

Transdermal compositions are typically formulated as a topical ointmentor cream containing the active ingredient(s), generally in an amountranging from about 0.01 to about 20% by weight, preferably from about0.1 to about 20% by weight, preferably from about 0.1 to about 10% byweight, and more preferably from about 0.5 to about 15% by weight. Whenformulated as a ointment, the active ingredients will typically becombined with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream with,for example an oil-in-water cream base. Such transdermal formulationsare well-known in the art and generally include additional ingredientsto enhance the dermal penetration of stability of the active ingredientsor the formulation. All such known transdermal formulations andingredients are included within the scope of this invention.

The compounds of this invention can also be administered by atransdermal device. Accordingly, transdermal administration can beaccomplished using a patch either of the reservoir or porous membranetype, or of a solid matrix variety.

The above-described components for orally administrable, injectable ortopically administrable compositions are merely representative. Othermaterials as well as processing techniques and the like are set forth inPart 8 of Remington's Pharmaceutical Sciences 17th edition, 1985, MackPublishing Company, Easton, Pa., which is incorporated herein byreference.

The compounds of this invention can also be administered in sustainedrelease forms or from sustained release drug delivery systems. Adescription of representative sustained release materials can be foundin Remington's Pharmaceutical Sciences.

The following examples illustrate representative pharmaceuticalcompositions that may be prepared in accordance with this invention. Thepresent invention, however, is not limited to the followingpharmaceutical compositions.

Formulation 1 Tablets

A compound of the invention may be admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnesium stearate is added as a lubricant. The mixture is formed into240-270 mg tablets (80-90 mg of active compound per tablet) in a tabletpress.

Formulation 2 Capsules

A compound of the invention may be admixed as dry powder with a starchdiluents in an approximately 1:1 weight ratio. The mixture is filledinto 250 mg capsules (125 mg of active compound per capsule).

Formulation 3 Liquid

A compound of the invention (125 mg) may be admixed with sucrose (1.75g) and xanthan gum (4 mg) and the resultant mixture may be blended,passed through a No. 10 mesh U.S. sieve, and then mixed with apreviously made solution of microcrystalline cellulose and sodiumcarboxymethyl cellulose (11:89, 50 mg) in water. Sodium benzoate (10mg), flavor, and color are diluted with water and added with stirring.Sufficient water may then be added to produce a total volume of 5 mL

Formulation 4 Tablets

A compound of the invention may be admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnesium stearate is added as a lubricant. The mixture is formed into450-900 mg tablets (150-300 mg of active compound) in a tablet press.

Formulation 5 Injection

A compound of the invention is dissolved or suspended in a bufferedsterile saline injectable aqueous medium to a concentration ofapproximately 5 mg/mL.

Formulation 6 Topical

Stearyl alcohol (250 g) and a white petrolatum (250 g) are melted atabout 75° C. and then a mixture of a compound of the invention (50 g)methylparaben (0.25 g), propylparaben (0.15 g), sodium lauryl sulfate(10 g), and propylene glycol (120 g) dissolved in water (about 370 g) isadded and the resulting mixture is stirred until it congeals.

Methods of Treatment

The present compounds are used as therapeutic agents for the treatmentof conditions in mammals that are causally related or attributable toaberrant activity of MMP1 and/or MAPKAPK5. Accordingly, the compoundsand pharmaceutical compositions of this invention find use astherapeutics for preventing and/or treating inflammatory diseases inmammals including humans.

In a method of treatment aspect, this invention provides a method oftreating a mammal susceptible to or afflicted with a conditionassociated with extra-cellular matrix (ECM) degradation, in particulararthritis, and more particularly, rheumatoid arthritis which methodcomprises administering an effective amount of one or more of thecompounds of the invention or a pharmaceutical composition as justdescribed.

In another method of treatment aspect, the invention provides a methodof treating a mammal susceptible to or afflicted with a conditionassociated with an abnormal cellular expression of MMP1, which comprisesadministering a therapeutically effective amount of a compound of theinvention, or a pharmaceutical composition thereof.

In another method of treatment aspect, the present invention provides amethod of treatment or prophylaxis of a condition characterized byabnormal matrix metallo proteinase activity, which comprisesadministering a therapeutically effective matrix metallo proteinaseinhibiting amount of one or more of the compounds of the invention, orpharmaceutical composition thereof.

In yet another method of treatment aspect, this invention providesmethods of treating a mammal susceptible to or afflicted with diseasesand disorders which are mediated by or result in inflammation such as,for example rheumatoid arthritis and osteoarthritis, myocardialinfarction, various autoimmune diseases and disorders, uveitis andatherosclerosis; itch/pruritus such as, for example psoriasis; and renaldisorders wherein said method comprises administering an effectivecondition-treating or condition-preventing amount of one or more of thecompounds of the invention or pharmaceutical compositions justdescribed.

This invention also relates to the use of the present compounds in themanufacture of a medicament for treatment or prophylaxis of a conditionprevented, ameliorated or eliminated by administration of an inhibitorof Mitogen-Activated Protein Kinase-Activated Protein Kinase 5, or acondition characterised by abnormal collagenase activity, or a conditionassociated with ECM degradation or a condition selected from diseasesinvolving inflammation, most preferably in for the treatment ofrheumatoid arthritis.

The invention also provides a compound of the invention for use in thetreatment of a condition associated with extra-cellular matrix (ECM)degradation, in particular arthritis, e.g. rheumatoid arthritis.

The invention also provides a compound of the invention for use in thetreatment of a condition associated with inflammation, such as, forexample rheumatoid arthritis and osteoarthritis, myocardial infarction,various autoimmune diseases and disorders, uveitis and atherosclerosis;itch/pruritus such as, for example psoriasis; and renal disorders.

The invention also provides a compound of the invention for use in thetreatment of a condition prevented, ameliorated or eliminated byadministration of an inhibitor of Mitogen-Activated ProteinKinase-Activated Protein Kinase 5, or a condition characterised byabnormal collagenase activity.

As a further aspect of the invention there is provided the presentcompounds for use as a pharmaceutical especially in the treatment orprevention of the aforementioned conditions and diseases. Also providedherein is the use of the present compounds in the manufacture of amedicament for the treatment or prevention of one of the aforementionedconditions and diseases.

A preferred regimen of the present method comprises the administrationto a subject in suffering from a disease condition characterized byinflammatory, with an effective matrix metallo-protease inhibitingamount of a compound of the present invention for a period of timesufficient to reduce the abnormal levels of extracellular matrixdegradation in the patient, and preferably terminate, theself-perpetuating processes responsible for said degradation. A specialembodiment of the method comprises administering of an effective matrixmetallo-protease inhibiting amount of a compound of the presentinvention to a subject patient suffering from or susceptible to thedevelopment of rheumatoid arthritis, for a period of time sufficient toreduce or prevent, respectively, collagen and bone degradation in thejoints of said patient, and preferably terminate, the self-perpetuatingprocesses responsible for said degradation.

In one embodiment, the treatment regimen involves intra-articularadministration of a compound of the invention or a pharmaceuticalcomposition as described herein.

Injection dose levels range from about 0.1 mg/kg/hour to at least 10mg/kg/hour, all for from about 1 to about 120 hours and especially 24 to96 hours. A preloading bolus of from about 0.1 mg/kg to about 10 mg/kgor more may also be administered to achieve adequate steady statelevels. The maximum total dose is not expected to exceed about 2 g/dayfor a 40 to 80 kg human patient.

For the prevention and/or treatment of long-term conditions, such asinflammatory and autoimmune conditions, the regimen for treatmentusually extends over many months or years, and accordingly oral dosingis preferred for patient convenience and tolerance. With oral dosing,one to five and especially two to four and typically three oral dosesper day are representative regimens. Using these dosing patterns, eachdose provides from about 0.01 to about 20 mg/kg of the compound of theinvention, with preferred doses each providing from about 0.1 to about10 mg/kg and especially about 1 to about 5 mg/kg.

Transdermal doses are generally selected to provide similar or lowerblood levels than are achieved using injection doses.

When used to prevent the onset of an inflammatory condition, thecompounds of this invention will be administered to a patient at riskfor developing the condition, typically on the advice and under thesupervision of a physician, at the dosage levels described above.Patients at risk for developing a particular condition generally includethose that have a family history of the condition, or those who havebeen identified by genetic testing or screening to be particularlysusceptible to developing the condition.

The compounds of this invention can be administered as the sole activeagent or they can be administered in combination with other agents,including other compounds that demonstrate the same or a similartherapeutic activity, and that are determined to safe and efficaciousfor such combined administration.

General Synthetic Procedures

The triazolopyrazine and imidazopyrazine compounds of this invention canbe prepared from readily available starting materials using thefollowing general methods and procedures. It will be appreciated thatwhere typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and P. G. M. Wuts, ProtectingGroups in Organic Synthesis, Second Edition, Wiley, New York, 1991, andreferences cited therein.

The following methods are presented with details as to the preparationof representative bicycloheteroaryls that have been listed hereinabove.The compounds of the invention may be prepared from known orcommercially available starting materials and reagents by one skilled inthe art of organic synthesis.

All reagents were of commercial grade and were used as received withoutfurther purification, unless otherwise stated. Commercially availableanhydrous solvents were used for reactions conducted under inertatmosphere. Reagent grade solvents were used in all other cases, unlessotherwise specified. Column chromatography was performed on silica gel60 (35-70 μm). Thin layer chromatography was carried out usingpre-coated silica gel F-254 plates (thickness 0.25 mm). ¹H NMR spectrawere recorded on a Bruker DPX 400 NMR spectrometer (400 MHz). Chemicalshifts (δ) for ¹H NMR spectra are reported in parts per million (ppm)relative to tetramethylsilane (δ 0.00) or the appropriate residualsolvent peak, i.e. CHCl₃ (δ 7.27), as internal reference. Multiplicitiesare given as singlet (s), doublet (d), triplet (t), quartet (q),multiplet (m) and broad (br). Coupling constants (J) are given in Hz.Electrospray MS spectra were obtained on a Micromass platform LC/MSspectrometer. Column Used for all LCMS analysis: Waters Acquity HPLC BEHC18 1.7 μm, 2.1 mm ID×50 mm L (Part No. 186002350)). Preparative HPLC:Waters XBridge Prep C18 5 μm ODB 19 mm ID×100 mm L (Part No. 186002978).All the methods are using MeCN/H₂O gradients. H₂O contains either 0.1%TFA or 0.1% NH₃.

List of Abbreviations Used in the Experimental Section:

DCM: Dichloromethane EtOAc ethyl acetate DiPEA:N,N-diisopropylethylamine APCI atmospheric pressure MeCN Acetonitrilechemical ionization BOC tert-Butyloxy-carbonyl Rt retention time DMFN,N-dimethylformamide s Singlet TFA Trifluoroacetic acid br s broadsinglet THF Tetrahydrofuran m Multiplet NMR Nuclear Magnetic d DoubletResonnance PdCl₂dppf [1,1′- DMSO Dimethylsulfoxide Bis- DPPADiphenylphosphorylazide (diphenylphosphino)- LC-MS LiquidChromatography- ferrocene] Mass Spectrometry dichloropalladium(II) Ppmpart-per-million TEA Triethylamine

Synthetic Preparation of Compounds of the Invention Synthesis ofIntermediates Intermediate 1a: Preparation of3,6-Dibromo-pyrazin-2-ylamine General Reaction Scheme:

Step 1: Synthesis of compound (B) as described in the general reactionscheme; 3,6-Dibromo-Pyrazine-2-Carboxylic Acid

LiOH (655 mg, 27 mmol) is added to a solution of3,6-dibromo-pyrazine-2-carboxylic acid methyl ester (A) (J. Med. Chem.1969, 12, 285-87) (2.7 g, 9 mmol) in THF:water:MeOH (18:4.5:4.5 mL). Thereaction is stirred at 5° C. for 30 min, concentrated in vacuo, taken upin DCM and washed with 1N HCl. The organic phase is dried over MgSO₄ andconcentrated in vacuo to afford compound (B). ¹H NMR (250 MHz, CDCl₃) δ(ppm) 8.70 (s, 1H).

Step 2: Synthesis of compound (C) as described in the general reactionscheme; 3,6-Dibromo-pyrazin-2-ylamine

Diphenylphosphorylazide (2.59 mL, 12 mmol) and triethylamine (1.67 mL,12 mmol) are added to a solution of 2,5-dibromo-3-pyrazoic acid (3.52 g,12 mmol) in t-butanol (90 mL). The reaction is heated at reflux for 18hours. The reaction is quenched with water, then concentrated in vacuoand taken up in DCM. The organic solution is washed with water and 1NNaOH, dried over MgSO₄ and concentrated in vacuo. The resultant solid isfiltered through a pad of silica using EtOAc, then concentrated andTFA:DCM (4:1, 12 mL) is added to the solid and stirred for 30 min. Thesolution is concentrated in vacuo then neutralised with 1N NaOH andextracted with DCM. The organic layer is dried over MgSO₄ andconcentrated in vacuo to give the product. ¹H NMR (250 MHz, d₆-DMSO) δ(ppm) 7.25 (br s, 2H), 7.68 (s, 1H); m/z (APCI) 254 (M+H)⁺; m.p 135-139°C.

Intermediate 1a: Preparation of 3,6-Dibromo-pyrazin-2-ylamine

Alternatively 3-Chloro-6-bromo-pyrazin-2-ylamine can be used and isprepared according to the following scheme:

Step 1. Synthesis of compound (A) as described in the general reactionscheme; 3,6-dibromo-2-chloropyrazine

To a well stirred solution of 2-chloro-3,6-dibromopyrazine (3.21 g,12.692 mmol) in DCM (20 mL) cooled to 0° C. is added TiCl₄ (2.41 g,12.692 mmol, 1.00 equiv.) in one portion, thus giving a dark red slurry.tButylnitrite (2.62 g, 25.385 mmol, 2.00 equiv.) is then added dropwise,causing the solution to turn bright yellow. The ice bath is then removedand the reaction is then allowed to proceed at room temperature. MoreTiCl₄ (1.50 g, 1.2 equiv.) is added and the mixture is stirred furtherfor one hour. At that point an orange solution has formed and LC-MSshows full conversion of the starting material to the desired productwhich ionises very poorly. Water (100 mL) is added to the reaction,forming an emulsion. DCM (50 mL) is added, and the DCM layer isseparated, and the aqueous layer is further extracted with DCM (3×50 mL)until the DCM layer is colorless. The DCM layers are gathered, washedwith brine and dried over Na₂SO₄, to yield after solvent removal,compound A′ (2.81 g, 82%) as an orange oil, which is used as such in thefollowing step.

Step 2: Synthesis of compound (B′) as described in the general reactionscheme; 2-Amino-3-chloro-6-bromopyrazine

Compound A′ described in the previous step (9.5 g, 37.55 mmol) issuspended in concentrated NH₄OH (60 mL) and the resulting mixture isheated in a pressure autoclave to 80° C., typically overnight. Thevessel is then allowed to cool down to room temperature slowly, and isthen further cooled in an ice bath, causing the precipitation of thedesired material. The solid is separated by filtration, washed withcyclohexane, to afford after drying, the title compound B′ (5 g) as a83/17 mixture of regioisomers. The mixture is then purified by columnchromatography. M+H+, m/z=209

Intermediate 2: 5,8-Dibromo-imidazo[1,2-a]pyrazine

Bromoacetaldehyde diethyl acetal (49 mL, 326 mmol) and 48% hydrobromicacid is heated to reflux for 1.5 h, then poured into propan-2-ol (600mL) and quenched with NaHCO₃. After filtering,2,5-dibromo-3-aminopyrazine (41.34 g, 163 mmol) is added to the solutionand heated at reflux overnight. The reaction is cooled and solventsremoved in vacuo, followed by addition of aq. NaHCO₃ and extraction withEtOAc. The organic phase is dried over MgSO₄, filtered, and concentratedin vacuo to afford a brown solid. ¹H NMR (250 MHz, CDCl₃) δ (ppm) 7.86(s, 1H), 7.93-7.94 (d, 1H), 7.98-7.99 (d, 1H); m/z (APCI) 278 (M+H)⁺;m.p 132-135° C.

Intermediate 3: 5,8-Dibromo-[1,2,4]-triazolo[1,5-a]pyrazine

General Scheme:

Step 1: N′-(3,6-Dibromo-pyrazin-2-yl)-N,N-dimethylformamidine(D)

A mixture of 3,6-dibromo-pyrazin-2-ylamine (15.37 g, 60.80 mmol) andN,N-dimethylformamide dimethyl acetal (10.1 mL, 76.00 mmol), suspendedin ethanol (150 mL), is refluxed for 2 hours. The reaction mixture isevaporated in vacuo affording the title compound. ¹H-NMR (400 MHz,CDCl₃) δ (ppm) 3.20 (s, 3H), 3.21 (s, 3H), 7.93 (s, 1H), 8.48 (s, 1H).LCMS: Rt 3.81 min (99.1%), m/z (APCI) 307 (M+H)⁺.

Step 2: N-(3,6-Dibromo-pyrazin-2-yl)-N′-hydroxyformamidine (E)

To a solution of N′-(3,6-dibromo-pyrazin-2-yl)-N,N-dimethylformamidine(18.6 g, 60.80 mmol) in methanol (200 mL) is added hydroxylaminehydrochloride (5.91 g, 85.12 mmol) in one portion. The reaction isstirred at room temperature for 16 hours. The solvent is evaporated andthe solid residue is treated with cold (ice cooling) water and collectedby filtration. The precipitate is washed twice with water and petroleumether and dried in vacuo yielding the title compound. ¹H-NMR (400 MHz,d₆-DMSO) δ (ppm) 7.82 (br s, 1H), 8.21 (s, 1H), 8.34 (m, 1H), 11.17 (brs, 1H). LCMS: Rt 3.17 min (98.7%), m/z (APCI) 295 (M+H)⁺.

Step 3: 5,8-Dibromo-[1,2,4]triazolo[1,5-a]pyrazine (F)

N-(3,6-dibromo-pyrazin-2-yl)-N′-hydroxyformamidine (17.4 mg, 58.80 mmol)is treated with polyphosphoric acid (150 g) for one hour at 50° C. andthen for 1.75 hours at 70° C. After cooling to room temperature, wateris added to the reaction mixture. The resultant suspension is brought topH 8 by careful addition of solid NaHCO₃ in small portions. Theprecipitate formed is collected by filtration, washed once with 1N NaOH,three times with water and dried in vacuo. The residue is partitionedbetween ethyl acetate and 1N NaOH and the organic phase is washed onemore time with 1N NaOH and once with brine. The organic phase is driedover MgSO₄, filtered and evaporated to give the title compound (10.15 g)as a white solid. ¹H-NMR (400 MHz, d₆-DMSO) δ (ppm) 8.43 (s, 1H), 8.92(s, 1H). LCMS: Rt 2.73 min (94.2%), m/z (APCI) 277 (M+H)⁺.

Intermediate 4:5-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-2,3-dihydro-isoindol-1-one

Step 1: 4-Bromo-2-bromomethyl-benzoic acid methyl ester

4-Bromo-2-methyl-benzoic acid (4.6 g, 21.39 mmol) is dissolved in 2M HClin MeOH and refluxed for 3 hours. The solvent is evaporated to give the4-bromo-2-methyl-benzoic acid methyl ester (4.24 g, 86%). Thisintermediate (18.51 mmol) is dissolved in carbon tetrachloride (100 mL)and N-bromosuccinimide (5.57 g, 24.06 mmol) is added. AIBN (122 mg, 740μmol) is then added and the mixture purged with nitrogen for 5 minutes.The reaction mixture is then refluxed for four hours. After cooling toroom temperature the reaction mixture is filtered and the filtrate isevaporated. The residue is purified by flash chromatography (silica gel,2:1 petroleum ether/ethyl acetate) to give the title compound (3.42 g,60%).

Step 2: 5-Bromo-2,3-dihydro-isoindol-1-one

4-Bromo-2-bromomethyl-benzoic acid methyl ester (0.5 g, 16.2 mmol) istreated with methanolic ammonia (10 mL, 7 N NH₃ in MeOH) for 5 minutesat 90° C. After cooling to room temperature the precipitate formed isfiltered off and washed with a small amount of methanol affording thetitle compound (224 mg, 65%) as a colourless solid.

¹H-NMR (400 MHz, d₆-DMSO) δ=4.41 (s, 2H, CH₂), 7.64 (d, 1H, H_(ar)),7.70 (d, 1H, H_(ar)), 7.87 (s, 1H, H_(ar)), 8.67 (bs, 1H, NH). LCMS99.6%, R_(t)=2.49 min, m/z 212 (M+H, AP⁺formic acid).

Step 3:5-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-2,3-dihydro-isoindol-1-one

5-Bromo-2,3-dihydro-isoindol-1-one (230 mg, 1.08 mmol),bis(pinacolato)diboron (300 mg, 1.18 mmol), Pda₂dppf (25 mg, 31 μmol)and KOAc (320 mg, 3.26 mmol) are suspended in dioxane (4 ml), purgedwith nitrogen for 5 minutes and then heated at 85° C. overnight. Thesolvent is removed in vacuo and the residue partitioned between ethylacetate and water. The aqueous layer is extracted three times with ethylacetate and the combined, organic phases are washed once with brine,filtered through MgSO₄ and evaporated. The solid residue is trituratedwith hexane and dried in vacuo to furnish the title compound (185 mg,66%) as a grey solid.

¹H-NMR (400 MHz, CDCl₃) δ=1.37 (s, 12H, 4×CH₃), 4.45 (s, 2H, CH₂), 6.38(bs, 1H, NH), 7.87 (d, 1H, H_(ar)), 7.93 (m, 2H, H_(ar)).

Intermediate 5:4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-furan-2-carboxylic acidamide

Step 1: 4-Bromo-furan-2-carboxylic acid amide

To a cooled (using a cold water bath) solution of4,5-dibromo-furan-2-carboxylic acid (12.5 g, 46.32 mmol) in NH₄OH (100mL) is added zinc dust (activated, powdered (washed with 2M HCl, water,MeOH, CH₂Cl₂) 4.54 g, 65.39 mmol) in small portions. The reactionmixture is stirred at room temperature for 10 minutes then filtered overcelite and washed with water. The filtrate is cooled to −10° C.(ice/salt bath) and acidified slowly to pH 1 using conc. HCl. The aqlayer is immediately extracted with ethyl acetate (4×). The organicphase is washed with brine, dried over MgSO₄, filtrated and concentratedin vacuo to give an oil (4.96 g) which solidifies on standing to give awhite solid, which is used without further purification.

The solid (4.93 g, 25.81 mmol) is dissolved in thionyl chloride (44.2mL) and refluxed for 1 hour. After removing the solvent in vacuo theresidue is dissolved in dichloromethane (75 mL) and a solution of 0.5 MNH₃ in dioxane (52 mL) is added. The reaction mixture is stirred at roomtemperature for 1 hour, then 33% aq. NH₃ (5 mL) is added and thereaction stirred for additional 2 hours. The solvent is removed in vacuoand the residue taken-up with a solution of sat. NaHCO₃. The basicsolution is extracted using ethyl acetate (3×), the combined organiclayers are dried over MgSO₄ and concentrated in vacuo. Purification bysilica gel column chromatography eluting with a mixture of (50:49:1)ethyl acetate: petroleum ether: acetic acid, affords the title compound(1.2 g, 22%).

Step 2:4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-furan-2-carboxylic acidamide

4-Bromo-furan-2-carboxylic acid amide (1.2 g, 6.32 mmol),bis(pinacolato)diboron (1.76 g, 6.94 mmol), PdCl₂dppf (0.154 g, 0.189mmol) and KOAc (1.85 g, 18.94 mmol) are suspended in dioxane (20 mL),purged with nitrogen for 5 minutes and then heated at 85° C. overnight.The solvent is removed in vacuo and the residue partitioned betweenethyl acetate and brine. The aqueous layer is extracted four times withethyl acetate, filtered through MgSO₄ and evaporated. The solid residueis triturated with hexane and dried in vacuo to afford the titlecompound as a solid (0.984 g, 66%). N.B. compound is usually 50-60% pureby ¹H-NMR.

Intermediate 6: 4(1-morpholinomethyl)thiazol-2-ylamine

Morpholine (350 μL, 4.00 mmol) is added to a solution of4-chloromethyl-thiazol-2-ylamine hydrochloride (368 mg, 2.00 mmol) inDMF (7 mL). The reaction is stirred at 90° C. for 16 h. The reaction isdiluted with ethyl acetate and washed with water. The aqueous layer isconcentrated in vacuo and extracted with ethyl acetate. The combinedorganic layers are dried over MgSO₄ and evaporated to give browncrystals which are used without further purification. ¹H-NMR (400 MHz,d₆-DMSO) δ=2.41-2.42 (m, 4H, CH₂CH₂), 3.30 (s, 2H, CH₂), 3.55-3.60 (m,4H, CH₂CH₂), 6.33 (s, 1H, H_(ar)), 6.85 (bs, 2H, NH₂).

Intermediate 7: 5-(tert-butyl-dimethylsilyloxymethyl)-thiazol-2-ylamine

A solution of 5-(hydroxymethyl)-thiazol-2-ylamine (0.787 g, 6.05 mmol),TBDMSCl (1.003 g, 6.65 mmol) and imidazole (0.49 g, 7.26 mmol) in DMF(15 mL) is stirred at room temperature for 24 h. The reaction mixture isdiluted with ether and water. The organic layer is washed with water(3×). The aqueous washings are backwashed with ether (2×). The organiclayers are combined, dried over MgSO₄ and condensed. The crude materialis purified using a plug of silica and eluting with 1:1 petrol:ethylacetate to give the product (0.656 g, 44%). ¹H-NMR (400 MHz, d₆-DMSO)δ=0.01 (s, 6H, 2×CH₃), 0.82 (s, 9H, tBu), 4.61 (s, 2H, CH₂), 4.74 (brs,2H, NH₂), 6.8 (s, 1H, CH). LCMS: Rt 2.62 min (92.7%). MS (MH⁺, m/z) 245.

Intermediate 8: (2-aminothiazol-5-yl)(morpholino)methanone

To a mixture of 5-(2-amino thiazole)-carboxylic acid (0.40 g, 2.77mmol), morpholine (0.241 mL, 2.77 mmol), HOBt (0.41 g, 3.05 mmol) andtriethylamine (0.77 mL, 5.54 mmol) in dichloromethane at 0° C. is addedEDCI (0.58 g, 3.05 mmol) and the reaction stirred at room temperature.After 3 h DMF (10 mL) is added to improve solubility and the reaction isheated at 30° C. overnight. The reaction is condensed then water addedto afford a precipitate. The mixture is cooled then filtered and theresulting solid product dried under vacuum (0.32 g, 54%). ¹H-NMR (400MHz, d₆-DMSO)S=3.6 (s, 8H, (CH₂CH₂)₂), 7.38 (s, 1H, H_(ar)), 7.48 (s,2H, NH₂). MS (MH⁺, m/z) 214.

Intermediate 9: 2-Amino-5-(4-morpholino methyl)thiazole

To a solution of morpholine (68 mg, 0.78 mmol) in methanol (3 mL) andacetic acid (0.045 mL, 0.78 mmol) is added sodium acetate (64 mg, 0.78mmol) followed by 2-amino-5-formyl thiazole (100 mg, 0.78 mmol). Thereaction is then heated to 40° C. for 1 h. Sodium cyanoborohydride (98mg, 1.56 mmol) is added and the reaction stirred at 40° C. overnight.The reaction is cooled, condensed and purified by column chromatography,eluting with 3% methanolic ammonia (7 N) in dichloromethane to give thetitle compound (91 mg, 58%). ¹H-NMR (400 MHz, CDCl₃) δ=2.451-2.47 (m,4H; CH₂CH₂), 3.68-3.71 (m, 4H, CH₂CH₂), 4.98 (s, 2H, CH₂), 6.87 (s, 1H,H_(a)). MS (MH⁺, m/z) 200.

Intermediate 10: 1-(2-morpholinoethyl)-1H-imidazol-2-amine Step 1:4-(2-(2-nitro-1H-imidazol-1-yl)ethyl)morpholine

2-Nitroimidazole (0.55 g, 4.86 mmol), 4-(2-chloroethyl)morpholinehydrochloride (0.90 g, 4.86 mmol), sodium iodide (0.18 g, 1.21 mmol) andsodium methoxide (0.59 g, 10.0 mmol) are dissolved in DMF (25 mL). Themixture is heated at 100° C. under a nitrogen atmosphere for 14 h. Thereaction mixture is cooled and solvent removed in vacuo. The residue ispartitioned between ethyl acetate (30 mL) and sat. NaHCO₃ (aq.) (30 mL).The aqueous layer is extracted with ethyl acetate (4×30 mL). The organiclayers are combined, dried (MgSO4) and solvent removed in vacuo. Thesolid residue is purified by column chromatography, eluting with 3%methanol in dichloromethane to give a yellow oil (638 mg, 58%). ¹H-NMR(400 MHz, d₆-DMSO) δ=2.39-2.45 (m, 4H), 2.66 (t, 2H, J=6 Hz), 3.52 (t,4H, J=4.8 Hz), 4.52-4.54 (t, 2H, J=6 Hz), 7.19 (s, 1H, H_(ar)), 7.68 (s,1H, H_(ar)).

Step 2: 1-(2-morpholinoethyl)-1H-imidazol-2-amine

The product from Step 1 (0.64 g, 2.81 mmol) is suspended in methanol (20mL) and water (2 mL). Palladium on carbon (0.065 g, 0.281 mmol) is addedand the mixture is degassed with nitrogen for 10 min before sodiumborohydride (0.15 g, 3.93 mmol) is added portion-wise. The reaction iscooled with an ice bath and after 30 mins warmed to room temperature andstirred for 16 h at room temperature. The palladium is filtered throughcelite and washed with methanol (3×30 mL). The filtrate is concentratedin vacuo and the residue partitioned between dichloromethane (100 mL)and water (10 mL). The aqueous layer is extracted with dichloromethane(5×50 mL+1 mL MeOH). The organic layers are combined, dried (MgSO₄) andsolvent removed in vacuo to give an orange oil (0.42 g, 77%). NMR (400MHz, CDCl3) δ=2.47-2.60 (m, 4H), 2.64-2.66 (m, 2H), 3.62-3.72 (m, 4H),3.77-3.83 (m, 2H), 4.76 (brs, 2H), 6.49 (s, H), 6.61 (s, 1H).

Example 15-(8-(4-(morpholinomethyl)thiazol-2-ylamino)imidazo[1,2-a]pyrazin-5-yl)isoindolin-1-oneStep 1: tert-butyl4-(5-bromo-[1,2,4]triazolo[1,5-a]pyrazin-8-ylamino)piperidine-1-carboxylate

Intermediate 2 (5,8-Dibromoimidazo[1,2-a]pyrazine, 177 mg, 0.63 mmol),Intermediate 6 (4-(1-morpholinomethyl)thiazol-2-ylamine, 140 mg, 0.70mmol), sodium tert-butoxide (86 mg, 0.89 mmol), palladium dibenzylideneacetone (23 mg, 0.026 mmol) and Xantphos (30 mg, 0.051 mmol) weresuspended in toluene (4 mL). The mixture is degassed with nitrogen for 5min and then stirred at 90° C. for 16 h. The solvent is evaporated invacuo then water is added and the resulting brown precipitate collected.The solid is washed with water then ether and dried in air. The solidresidue is purified by column chromatography, eluting with 2% methanolicammonia (7 N) in dichloromethane to give a yellow powder (82 mg, 34%).¹H-NMR (400 MHz, d₆-DMSO) δ=2.47 (s, 4H, CH₂CH₂), 3.53 (s, 2H, CH₂),3.62 (s, 4H, CH₂CH₂), 6.99 (s, 1H, H_(ar)), 7.85 (s, 1H, H_(ar)), 8.16(s, 1H, H_(ar)), 11.79 (s, 1H, NH). MS (MH⁺, m/z) 395.

Step 2:5-(8-(4-(morpholinomethyl)thiazol-2-ylamino)imidazo[1,2-a]pyrazin-5-yl)isoindolin-1-one

The product of Step 1 (35 mg, 0.09 mmol), Intermediate 4(5-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-2,3-dihydro-isoindol-1-one,29 mg, 0.11 mmol), tetrakis palladium triphenylphosphine (26 mg, 0.02mmol), sodium carbonate solution (1.5 M, 0.472 mL, 0.71 mmol) anddioxane (2.8 mL) are combined in a flask. The mixture is degassed withnitrogen for 10 min then sealed and heated at 90° C. for 16 h. Brine isadded and the mixture is washed with dichloromethane followed by ethylacetate. The combined organic layers are dried over MgSO₄ and evaporatedin vacuo to afford a brown oil. The crude material is purified by columnchromatography, eluting with 3% methanolic ammonia (7 N) indichloromethane to give the title compound as a yellow powder (16 mg,40%). (400 MHz, d₆-DMSO) δ=2.49 (s, 4H, CH₂CH₂), 3.55 (s, 2H, CH₂), 3.63(s, 4H, CH₂CH₂), 7.00 (s, 1H, H_(ar)), 7.72 (s, 1H, H_(ar)), 7.81 (s,1H, H_(ar)), 7.88 (s, 2H, H_(ar)), 8.00 (s, 1H, H_(ar)), 8.10 (s, 1H,H_(ar)), 8.74 (s, 1H, NH), 11.65 (s br, 1H, NH). LCMS: Rt 8.04 min(92.2%). MS (MH⁺, m/z) 448.

Example 25-(8-(5-(hydroxymethyl)thiazol-2-ylamino)imidazo[1,2-a]pyrazin-5-yl)isoindolin-1-oneStep 1:N-(5-bromoimidazo[1,2-a]pyrazin-8-yl)-5-((tert-butyldimethylsilyloxy)methyl)thiazol-2-amine

Prepared from Intermediate 2 and Intermediate 7 using the proceduredescribed in Example 1, Step1. MS (MH⁺, m/z) 440.

Step 2: (2-(5-bromoimidazo[7,2-a]pyrazin-8-ylamino)thiazol-5-yl)methanol

The product of Step 1 (0.125 g, 0.28 mmol) is dissolved in THF (2 mL).To this solution is added TBAF solution (1M in THF, 0.31 mL, 0.31 mmol)and the reaction is stirred at room temperature overnight. The reactionis condensed and the residue purified by gradient column chromatographyeluting with dichloromethane—5% methanolic ammonia (7 N) indichloromethane to give the alcohol (64 mg, 69%). LCMS: Rt 2.54 min(97.7%). MS (MH⁺, m/z) 326.

Step 3:5-(8-(5-(hydroxymethyl)thiazol-2-ylamino)imidazo[1.2-a]pyrazin-5-yl)isoindolin-1-one

Prepared from the product of Step 2 and Intermediate 4(5-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-2,3-dihydro-isoindol-1-one)according to the procedure described in Example 1, Step 2. ¹H-NMR (400MHz, d₆-DMSO) δ=4.52 (s, 2H, CH₂), 4.67 (d, 2H, CH₂), 5.40 (t, 1H, OH),7.36 (s, 1H, H_(ar)), 7.71 (s, 1H, H_(ar)), 7.81 (s, 1H, H_(ar)), 7.89(m, 2H, H_(ar)), 7.99 (s, 1H, H_(ar)), 8.09 (s, 1H, H_(ar)), 8.79 (s,1H, H), 11.61 (s br, 1H, NH). LCMS: Rt 2.04 min (90.0%). MS (MH⁺, m/z)379.

Example 3N-(5-(1H-pyrazol-4-yl)[1,2,4]triazolo[1,5-a]pyrazin-8-yl)-4-(morpholinomethyl)thiazol-2-amineStep 1:N-(5-bromo-[1,2,4]triazolo[1,5-a]pyrazin-8-yl)-4-(morpholinomethyl)thiazol-2-amine

Intermediate 3 (5,8-Dibromo-[1,2,4]triazolo[1,5-a]pyrazine, 140 mg, 0.50mmol), Intermediate 6 (4-(1-morpholinomethyl)thiazol-2-ylamine, 120 mg,0.60 mmol), sodium tert-butoxide (68 mg, 0.71 mmol), palladiumdibenzylidene acetone (19 mg, 0.02 mmol) and Xantphos (23 mg, 0.04 mmol)were suspended in toluene (4 mL). The mixture is degassed with nitrogenfor 5 min and then stirred at 90° C. for 16 h. The solvent is evaporatedin vacuo to leave a brown residue. The residue is purified by columnchromatography, eluting with 2% methanolic ammonia (7 N) indichloromethane to give the title, compound as a yellow powder (53 mg,27%). ¹H NMR (400 MHz; d₆-DMSO) δ 2.53 (s, 4H, CH₂CH₂), 3.51 (s, 2H,CH₂), 3.61 (m, 4H, CH₂CH₂), 6.99 (s, 1H), 8.13 (s, 1H), 8.61 (s, 1H),8.75 (s, 1H).

Step 2:N-(5-(1H-pyrazol-4-yl)-[1,2,4]triazolo[1,5-a]pyrazin-8-yl)-4-(morpholinomethyl)thiazol-2-amine

The product of Step 1 (43 mg, 0.11 mmol), 4-pyrazole boronic acidpinacol ester (32 mg, 0.16 mmol), tetrakis palladium triphenylphosphine(13 mg, 0.11 mmol) and sodium tert-butoxide are suspended in a mixtureof DMF (2 mL) and water (0.66 mL). The reaction is degassed withnitrogen for 10 min before being sealed and stirred at 90° C. for 16 h.The reaction is condensed in vacuo and the residue purified by columnchromatography, eluting with 3% methanolic ammonia (7 N) indichloromethane to give the title compound as a pale yellow powder (12mg, 29%). ¹H NMR (400 MHz, d₆-DMSO) δ 2.48 (s, 4H, CH₂CH₂), 3.35 (s, 2H,CH₂), 3.63 (t, 4H, J=6 Hz, CH₂CH₂), 6.97 (s, 1H, H), 8.43 (s, 1H, H),8.61 (brs, 2H), 8.81 (s, 1H, H), 12.9-12.4 (brs, 2H, NH×2). MS (MH⁺,m/z) 384.

Example 44-(8-(4-(piperidin-1-ylmethyl)thiazol-2-ylamino)-[1,2,4]triazolo[1,5-a]pyrazin-5-yl)furan-2-carboxamideStep 1:N-(5-bromo-[1,2,4]triazolo[1,5-a]pyrazin-8-yl)-4-(piperidin-1-ylmethyl)thiazol-2-amine

Prepared in a manner analogous to the procedure of Example 1, Step 1.NMR (400 MHz, d₆-DMSO) δ 1.42 (s, 2H), 1.53-1.55 (m, 4H), 2.37 (brs,4H), 3.77 (s, 2H), 6.93 (s, 1H), 8.11 (s, 1H), 8.72 (s, 1H). MS (MH⁺,m/z) 394.

Step 2:4-(8-(4-(piperidin-1-ylmethyl)thiazol-2-ylamino)-1,2,41-triazolo[1,5-a]pyrazin-5-ylfuran-2-carboxamide

The product of Step 1 (950 mg, 2.4 mmol), Intermediate 5 (4-(2-amido)furanyl boronic acid pinacol ester, 1.14 g, 4.8 mmol), tetrakispalladium triphenylphosphine (832 mg, 0.72 mmol), sodium carbonatesolution (1.5 M, 12.8 mL, 19.2 mmol) and dioxane (15 mL) are combined ina flask. The mixture is degassed with nitrogen for 10 min then sealedand heated at 90° C. for 16 h. The solvent is evaporated to give a crudematerial which is purified by gradient column chromatography, elutingwith 1-5% methanolic ammonia (7 N) in dichloromethane. The resultingyellow solid is triturated with 1:1 petrol:ether then 2:1dichloromethane:ether to give the title compound (346 mg, 29%). NMR (400MHz, d₆-DMSO) δ 1.43 (m, 2H, CH₂), 1.55 (m, 4H, CH₂CH₂), 2.45 (brs, 4H,CH₂CH₂), 3.50 (s, 2H, CH₂), 6.93 (s, 1H, H), 7.62 (s, 1H, H), 7.99 (s,2H, H), 8.44 (s, 1H, H), 8.82 (d, 2H, J=5.2 Hz, H). LCMS: Rt 2.00 min(95.7%). MS (MH⁺, m/z) 425.

Example 54-(8-(1-(2-morpholinoethyl)-1H-imidazol-2-ylamino)-[1,2,4]-triazolo[1,5-a]pyrazin-5-yl)furan-2-carboxamideStep 1:5-bromo-N-(1-(2-morpholinoethyl)-1H-imidazol-2-yl)-[1,2,4]triazolo[1,5-a]pyrazin-8-amine

Prepared using Intermediate 3 and Intermediate 10 in a manner analogousto the procedure of Example 1, Step 1. NMR (400 MHz, CDCl3) δ 2.55 (brs,4H), 2.71-2.80 (m, 2H), 3.68-3.83 (m, 4H), 4.15-4.26 (m, 2H), 6.92 (s,H), 7.05 (s, H), 7.71 (s, H), 8.31 (s, H), 8.58 (s, 1H).

Step 2:4-(8-(1-(2-morpholinoethyl)-1H-imidazol-2-ylamino)-[1,2,4]triazolo[1,5-a]pyrazin-5-yl)furan-2-carboxamide

Prepared in a manner analogous to the procedure of Example 3, Step 2.1HNMR (400 MHz, CDCl3) δ 2.54 (brs, 4H), 2.67-2.74 (m, 2H), 3.51-3.60 (m,4H), 4.17 (brs, 2H), 6.95 (s, H), 7.13 (s, 1H), 7.58 (s, 1H), 7.80 (s,H), 7.94 (s, H), 8.01 (s, H), 8.17 (s, H), 8.55 (s, H), 8.68 (s, H).LCMS: Rt 2.37 min (93.0%). MS (MH⁺, m/z) 424.

Examples 1-22 in Table 1 are made from the commercially available aminothiazoles and amino thiadiazoles and the Intermediates described above,or by procedures analogous to those described above.

TABLE 1 M + H+, Ex# Structure Name MW m/z 1

5-(8-(4- (morpholinomethyl)thiazol-2- ylamino)imidazo[1,2-a]pyrazin-5-yl)isoindolin-1-one 447.15 448 2

5-(8-(5-(hydroxymethyl)thiazol- 2-ylamino)imidazo[1,2-a]pyrazin-5-yl)isoindolin-1-one 482.22 379 3

N-(5-(1H-pyrazol-4-yl)- [1,2,4]triazolo[1,5-a]pyrazin-8- yl)-4-(morpholinomethyl)thiazol-2- amine 383.13 384 4

4-(8-(4-(piperidin-1- ylmethyl)thiazol-2-ylamino)-[1,2,4]triazolo[1,5-a]pyrazin-5- yl)furan-2-carboxamide 424.14 425 5

4-(8-(1-(2-morpholinoethyl)-1H- imidazol-2-ylamino)-[1,2,4]triazolo[1,5-a]pyrazin-5- yl)furan-2-carboxamide 423.18 424 6

5-(8-(thiazol-2- ylamino)imidazo[1,2-a]pyrazin-5- yl)isoindolin-1-one348.08 349 7

N-(5-(1H-pyrazol-4- yl)imidazo[1,2-a]pyrazin-8- yl)thiazol-2-amine283.06 284 8

5-(8-(5-(ethylsulfonyl)thiazol-2- ylamino)imidazo[1,2-a]pyrazin-5-yl)isoindolin-1-one 440.07 441 9

5-(8-(4-(piperidin-1- ylmethyl)thiazol-2-ylamino)imidazo[1,2-a]pyrazin-5- yl)isoindolin-1-one 445.17 446 10

N,N,4-trimethyl-2-(5-(1- oxoisoindolin-5-yl)imidazo[1,2-a]pyrazin-8-ylamino)thiazole-5- carboxamide 433.13 434 11

5-(8-(5-methylthiazol-2- ylamino)imidazo[1,2-a]pyrazin-5-yl)isoindolin-1-one 362.09 363 12

5-(8-(5,6-dihydro-4H- cyclopenta[d]thiazol-2-ylamino)imidazo[1,2-a]pyrazin-5- yl)isoindolin-1-one 388.11 389 13

5-(8-(5-(morpholine-4- carbonyl)thiazol-2-ylamino)imidazol[1,2-a]pyrazin-5- yl)isoindolin-1-one 461.13 462 14

5-(8-(5-(2-(piperidin-1-yl)ethyl)- 1,3,4-thiadiazol-2-ylamino)imidazo[1,2-a]pyrazin-5- yl)isoindolin-1-one 460.18 461 15

N-(5-(1H-pyrazol-4- yl)imidazo[1,2-a]pyrazin-8-yl)-5-(morpholinomethyl)thiazol-2- amine 382.13 383 16

5-(8-(5- (morpholinomethyl)thiazol-2- ylamino)imidazo[1,2-a]pyrazin-5-yl)isoindolin-1-one 447.15 448 17

N-(5-(1H-pyrazol-4- yl)imidazo[1,2-a]pyrazin-8-yl)-5-((dimethylamino)methyl)thiazol- 2-amine 340.12 341 18

5-(8-(5-((4-isopropylpiperazin-1- yl)methyl)thiazol-2-ylamino)imidazo[1,2-a]pyrazin-5- yl)isoindolin-1-one 488.21 489 19

5-(8-(1,3,4-thiadiazol-2- ylamino)imidazo[1,2-a]pyrazin-5-yl)isoindolin-1-one 349.07 350 20

5-(8-(5-(4-methylpiperazin-1-yl)- 1,3,4-thiadiazol-2-ylamino)imidazo[1,2-a]pyrazin-5- yl)isoindolin-1-one 447.16 448 21

(2-(5-(1H-pyrazol-4- yl)imidazo[1,2-a]pyrazin-8- ylamino)thiazol-5-yl)(morpholino)methanone 396.11 397 22

(2-(5-(1H-pyrazol-4- yl)imidazo[1,2-a]pyrazin-8-ylamino)thiazol-5-yl)(piperidin- 1-yl)methanone 394.13 395 23

(2-(5-(1H-pyrazol-4- yl)imidazo[1,2-a]pyrazin-5-ylamino)thiazol-5-yl)(4- isopropylpiperazin-1- yl)methanone 437.17 43824

5-(8-(5-(piperidin-1- ylmethyl)thiazol-2-ylamino)imidazo[1,2-a]pyrazin-5- yl)isoindolin-1-one 445.17 446 25

N-(5-(1H-pyrazol-4-yl)- [1,2,4]triazolo[1,5-a]pyrazin-8-yl)-4-(piperidin-1- ylmethyl)thiazol-2-amine 381.15 425

Purification Conditions and Characterization

Routinely, post-synthesis all compounds may be purified using reversephase HPLC using a Gilson preparative HPLC system (322 pump, 155 UV/VISdetector, 215 liquid handler). The Gilson 215 acts as both auto-samplerand fraction collector. Compounds can also be purified by flashchromatography on silica gel.

Compounds are characterised by mass spectrometry using single quadrupoleinstrumentation with an electrospray source.

Biological Assays Demonstrating the Usefulness of the Compounds Example1 MAPKAP-K5 Assay

MAPKAP-K5 reactions are performed in FlashPlate format using 0.1 or 0.2μCi 33P-ATP; 0.6 μM ATP; 1mU MAPKAP-K5; 3 μM MAPKAP-K5 peptidesubstrate, incubated at room temperature for 30 minutes.

Flashplate Assay:

The MAPKAP-K5 kinase reaction is performed in a 384 well polypropyleneplate (Matrix Technologies) and then transferred to astreptavidin-coated 384 well flashplate (Perkin-Elmer). To wellscontaining 2 μL test compound or standard inhibitor, 13 μL Enzyme mix ordiluent are added using a Hydra (Robbins Scientific). Reactions arestarted by addition of 10 μL of [2,5x] substrate cocktail using aMultidrop (Thermo-Labsystems), to give final concentrations in the assayof:

1 mU MAPKAP-K5

3 μM MAPKAP-K5 peptide substrate

˜0.6 μM ATP

0.004 μCi [33P]-γ-ATP/μL

1× reaction buffer

Plates are incubated at room temperature for 30 minutes. Reactions areterminated by the addition of 254, EDTA (50 mM) to each well using aMicro-fill (Biotek). Reactions are transferred to a streptavidin-coatedflashplate using a Zymark robotic system. Plates are incubated for 60minutes at room temperature. All wells are washed 3 times with 100 μLphosphate buffered saline using a Tecan plate washer. Radioactivity isdetermined by scintillation counting of the flashplate (empty wells) ona Packard TopCount.

Enzyme Mix:

Enzyme

50 mM Tris Hcl (pH 7.5)

0.1 mM EGTA

2 mM DTT

1 mg/ml BSA

Reaction Buffer:

50 mM Tris Hcl (pH 7.5)

0.1 mM EGTA

10 mM Magnesium acetate

2 mM DTT

Example 2 Development of an Assay for the Identification of Regulatorsof the Expression of MMP1 by Activated Primary Synovial Fibroblasts

To identify compounds that decrease the ECM-degrading activity of cells,the ECM-degrading activity of cells may be induced to allow properdetection of this activity, and to achieve a clearer read-out. In thecontext of RA, the cells of choice are mammalian synovial fibroblastsand the triggers that may be used to induce the ECM-degrading activityare cytokines relevant in the field of arthritis: for instance TNF-α,IL1β, IL6, OSM, IL17, and MIF1-α. This list is not comprehensive due tothe plethora of cytokines potentially involved in the RA pathogenesis(Smolen and Steiner, 2003). To set up an in vitro assay that is as closeas possible to the complexity of the pathology, the trigger appliedshould be a mixture of factors generated by contactingcytokine-producing cells relevant in the field of arthritis, such asmonocytes, macrophages, T-cells, and B-cells, with a trigger. Thecytokine-producing cells will respond to the contact by producing acomplex and unbiased mixture of factors. If the cytokine-producing cellused is also found in a pannus, and the cytokine applied to produce thistrigger is found in the synovial fluid of rheumatoid arthritis patients,the mixture of factors ultimately produced will contain part of thefactors that are present in the joints of arthritis patients.

Principle of the ‘MMP Assay’

Matrix Metallo Proteases (MMPs) possess various physiological roles, ase.g. the maturation of other proteases, growth factors, and thedegradation of extra-cellular matrix components. MMP1 is one of themembers of the MMP family that is able to degrade native collagen, themain component of bone and cartilage. An increased expression of MMP1 bysynovial fibroblasts (SFs) is diagnostic for the progression of thearthritic disease and is predictive for erosive processes in the joint(Cunnane et al., 2001). The expression of MMP1 by SFs can be increasedby the activation of SFs with triggers relevant for rheumatoidarthritis, as cytokines like TNF-α or IL1β (Andreakos et al., 2003).Taken together, measurement of the levels of MMP1 produced by activatedSFs is a readout that is highly relevant in the context of RA as thisevent reflects the level of activation of SFs towards an erosivephenotype as it is seen in the pannus. If a reduced expression of acandidate drug target in activated SFs leads to the reduction of MMP1expression by these cells, the drug target is then proven to be involvedin the regulation of MMP1 expression and thus considered relevant forthe development of therapeutic strategies for the treatment of RA.

In the following examples, the development of an assay, further referredto as ‘MMP assay’, monitors the MMP1 production by synovial fibroblasts(SFs) in response to diverse activating triggers (Example 2.1). The useof this assay is then described for the validation of gene products thatare considered drug targets for the development of RA therapies (Example2.2). The validation of drug targets is performed using recombinantadenoviruses, further referred to as knock-down viruses or Ad-siRNAs,that mediate the expression in cells of shRNA's which reduce theexpression levels of targeted genes by a RNAi (RNA interference)-basedmechanism (see WO 03/020931). The identification of compounds modulatingthe activity of the validated drug targets is then described in Table B.The use of the ‘MMP assay’ for the testing of compounds that modulatethe activity of the drug targets identified is described further below.

Control Viruses Used:

The control viruses used in these studies are listed below. dE1/dE2Aadenoviruses are generated from these adapter plasmids byco-transfection of the helper plasmid pWEAd5AflII-rITR.dE2A in PER.E2Apackaging cells, as described in WO99/64582.

Negative Control Viruses:

Ad5-eGFP_KD: Target sequence: GCTGACCCTGAAGTTCATC (SEQ ID NO: 1). Clonedusing Sap1-sites into vector and virus generated as described inWO03/020931.Ad5-Luc_v13_KD: Target sequence GGTTACCTAAGGGTGTGGC (SEQ ID NO: 2).Cloned using Sap1-sites into vector and virus generated as described inWO03/020931.Ad5-M6PR_v1_KD: Target sequence CTCTGAGTGCAGTGAAATC (SEQ ID NO: 3).Cloned using Sap1-sites into vector and virus generated as described inWO03/020931.

Positive Control Viruses:

Ad5-MMP1_v10_KD: Target sequence ACAAGAGCAAGATGTGGAC (SEQ ID NO: 4).Cloned using Sap1-sites into vector and virus generated as described inWO03/020931.

Viruses Used for Target Validation:

Ad5-MAPKAPK5_v13_KD: Target sequence CGGCACTTTACAGAGAAGC (SEQ ID NO: 5).Cloned using Sap1-sites into vector and virus generated as described inWO03/020931.Ad5-MAPKAPK5_v12_KD: Target sequence ATGATGTGTGCCACACACC (SEQ ID NO: 6).Cloned using Sap1-sites into vector and virus generated as described inWO03/020931.

Example 2.1 Development of the MMP Assay

A 384-well format ELISA for measurement of MMP1 is developed. Variousprimary antibodies are tested, as well as various ELISA protocols. Thefollowing protocol is developed and validated to measure MMP1 levels inSF supernatant in 384 well plates: white Lumitrac 600 384 well plates(Greiner) are coated with 2 μg/mL anti-MMP1 antibody MAB1346 (Chemicon).The antibody is diluted in buffer 40 (1.21 g Tris base (Sigma), 0.58 gNaCl (Calbiochem) and 5 ml 10% NaN₃ (Sigma) in 1 L milliQ water andadjusted to pH 8.5). After overnight incubation at 4° C., plates arewashed with PBS (80 g NaCl, 2 g KCl (Sigma), 11.5 g Na₂HPO₄.7H₂O and 2 gKH₂PO₄ in 10 L milliQ; pH 7.4) and blocked with 100 μL/well Caseinbuffer (2% Casein (VWR International) in PBS). Next day, casein bufferis removed from ELISA plates and replaced by 50 μL/well EC buffer (4 gcasein, 2.13 g Na₂HPO₄ (Sigma), 2 g bovine albumin (Sigma), 0.69 gNaH₂PO₄.H₂O (Sigma), 0.5 g CHAPS (Roche), 23.3 g NaCl, 4 mL 0.5 M EDTApH 8 (Invitrogen), 5 mL 10% NaN₃ in 1 L milliQ and adjusted to pH 7.0).0.25 mM DTT (Sigma) is added to the thawed samples plates. After removalof the EC buffer, 20 μL of sample is transferred to the ELISA plates.After overnight incubation at 4° C. plates are washed twice with PBS andonce with PBST (PBS with 0.05% Tween-20 (Sigma)) and incubated with 35μL/well biotinylated anti-MMP1 antibody solution (R&D). This secondaryantibody is diluted in buffer C (0.82 g NaH₂PO₄.H₂O, 4.82 g Na₂HPO₄,46.6 g NaCl, 20 g bovine albumin and 4 mL 0.5M EDTA pH 8 in 2 L milliQand adjusted to pH 7.0) at a concentration of 5 μg/mL. After 2 h ofincubation at RT, plates are washed as described above and incubatedwith 50 μL/well streptavidin-HRP conjugate (Biosource). Streptavidin-HRPconjugate is diluted in buffer C at a concentration of 0.25 μg/mL. After45 min, plates are washed as described above and incubated for 5 minwith 50 μL/well BM Chem ELISA Substrate (Roche). Readout is performed onthe Luminoscan Ascent Luminometer (Labsystems) with an integration timeof 200 msec or with an Envision reader (Perkin Elmer).

The increase of MMP1 expression by SFs upon treatment with cytokinesrelevant in the field of RA (TNF-α, IL1β and OSM) or a combinationthereof is shown in FIG. 2 as white bars. For this experiment, SFs areseeded in 96 well plates, 3,000 cells/well. 24 h later, the medium ischanged to M199 medium supplemented with 1% FBS. One day after themedium change, cytokines or combinations thereof are added to thecultures, each cytokine being added to a final concentration of 25ng/mL. 72 h after cytokine addition, the supernatant is collected andprocessed in the MMP1 ELISA as described in the protocol given above. Inparallel with this experiment, SFs are triggered, using the sameprotocol, with the supernatant of THP1 cells (2-fold diluted in M199+1%FBS) treated with the same cytokines or combinations of cytokines for 48h in M199 medium+1% FBS. MMP1 levels for these samples are shown in FIG.2 as grey bars. The induction of the MMP1 expression by SFs triggeredwith the supernatants of TNF-α-treated THP1 cells is stronger (>4.5 foldinduction) as compared to the SFs triggered with recombinant TNF-α alone(3-fold induction) and almost equals the 5-fold induction obtained by amixture of 3 purified cytokines (TNF-α, IL1βb, OSM). This resultindicates that the supernatant of TNF-α-induced THP1 cells contains,besides TNF-α, additional pro-inflammatory factors that activate SFstowards MMP1 expression. As the role of TNF-α in the RA pathogenesis isvalidated (TNF-α-blockers such as Infliximab and Etanercept show someefficacy in the treatment of RA patients) and the THP-1 cells arerepresentative for monocytes/macrophages present in the joint of RApatients, the TNF-α-based trigger mixture prepared by contacting THP-1cells with TNF-α will contain factors present in the joints of RApatients and subsequently is relevant to RA. This TNF-α-based complextrigger, further referred to as the ‘complex trigger’, will further beused as basis for the ‘MMP assay’.

Inhibition of the activation of SF by the ‘complex trigger’ is shownusing dexamethasone, a potent anti-inflammatory agent that also stronglyreduces collagen-induced arthritis in rodents (Yang et al., 2004) (FIG.3). Dexamethasone is shown to dose-dependently reduce amounts of MMP1produced by complex trigger activated SFs. SFs are seeded at a densityof 3000 cells/well in 96 well plates. 24 hrs after seeding, increasingconcentrations of dexamethasone are added to the cells. After overnightincubation, medium of every well is refreshed to supernatant of THP-1cells treated with TNF-α (50% diluted in M199+0.5% FBS), and the sameconcentration of dexamethasone as added the day before. 48 hrs aftertreatment, the supernatant is collected and subjected to the MMP1 ELISAdescribed above. The addition of dexamethasone clearly reduced the MMP1expression by SFs, with an IC₅₀ value of about 1 nM (see FIG. 3). Thesedata show that the MMP1 expression by activated SFs can be reduced bythe addition of a physiologically relevant inhibitor and represent aproof of principle for the ‘MMP assay’.

Example 2.2 MAPKAPK5 Modulates SF ‘Complex Trigger’-Induced MMP1Expression (A) Ad-siRNA Virus Functions to Knock Down MAPKAPK5Expression.

Recombinant adenoviruses mediating the expression of siRNA's targetingMAPKAPK5 and eGFP are generated according to the procedure described inWO03/020931. The target sequence used in the recombinant adenovirus is:CGGCACTTTACAGAGAAGC (SEQ ID NO: 5) as well as ATGATGTGTGCCACACACC (SEQID NO: 6). The target sequence within the eGFP mRNA used in therecombinant adenovirus is: GCTGACCCTGAAGTTCATC (SEQ ID NO: 1). Thesesequences are cloned into the adapter plasmid using Sap1 sites. dE1/dE2Aadenoviruses are generated from these adapter plasmids byco-transfection of the helper plasmid pWEAd5AflII-rITR.dE2A in PER.E2Apackaging cells, as described in WO99/64582.

The functionality of an adenovirus targeting MAPKAPK5 is tested asfollows. These adenoviruses are used to infect primary human SFscultured in petri dishes as follows. On day 1,500.000 SFs are seeded perpetri dish. One day later, the cells are infected withAd5-MAPKAPK5-v13_KD (1.6E9 VP/mL) or Ad5-eGFP-v5_KD (1.3E10 VP/mL) at anMOI of 4000 (based on the titers (number of virus particles per mL)defined for the viruses by Q-rt-PCR). On day 7, cells are detached fromthe petri dish according to standard procedure using a trypsin EDTAsolution. The trypsin is then neutralized by addition of DMEM growthmedium supplemented with 10% FBS. The cells are then collected by acentrifugation step (1000 rpm, 5 min). The pellet is lysed in 100 μL offresh RIPA buffer (50 mM Tris pH7.5, 150 mM NaCl, 1% deoxycholate, 1%Triton X100, 0.1% SDS). The samples are then sonicated for 10 sec. Theprotein concentration of the samples is then determined using the BCAkit (Pierce, Cat N^(o) 23227) as described by the provider, using BSA asa standard. To 30 μg of cell lysate diluted to 19.5 μl in RIPA buffer,3.5 μL of reducing agent (NuPage reducing agent N^(o)10, InvitrogenNP0004) and 7.5 μL of sample buffer (NuPage LDS sample buffer,Invitrogen NP0007) are added. The 30 μL sample is then boiled for 5 minand loaded on a 10% polyacrylamide gel (Invitrogen NP0301). To allow theestimation of the level of protein knock-down, 15 μg, 7.5 μg and 3.75 μgof the lysate of the Ad5-eGFP-v5_KD infected cells are also loaded ontothe gel. The gel is then run for 2 hours at 100V in 1×MOPS/SDS NuPagerunning buffer (Invitrogen NP001). 10 μl of Seablue Plus Prestainedstandard (Invitrogen LC5925) is used to estimate protein size on thegel. The proteins on the gel are then transferred onto a PVDF membrane(Invitrogen LC2002) by a wet blotting procedure using a transfer bufferprepared by mixing 100 ml Nupage Transfer buffer 20* (NP0006-1), 400 mLmethanol and 1500 mL Milli Q water. Before the transfer, the membrane isfirst soaked in methanol and in transfer buffer. The transfer isperformed at 100V for 90 minutes. The membrane is then blocked by 30 minsoaking in blocking buffer (2% blocking blocking powder (Amersham, RPN2109) prepared in PBST (PBS supplemented with 0.1% Tween 20 (Sigma,P1379)). After blocking, the immunodetection is performed using a mousemonoclonal antibody against MAPKAPK5 (BD Biosciences, Cat N^(o)612080)diluted 250 fold in blocking buffer. After overnight incubation withthis primary antibody, the membrane is washed 3 times with PBST andincubated 1 hr with the secondary antibody ((Polyclonal goat anti-mouseIg, I-IRP conjugated (DAKO P0447) diluted 50000 fold in blocking buffer.The blot is then washed 3 times in PBST and the detection is performedwith ECL advance (RPN2109, Amersham) on a Kodakimager according to themanufacturers instructions. The Western Blotting revealed a lowerexpression level of MAPKAPK5 in the Ad5-MAPKAPK5-v13_KD infected cellscompared to the cells infected with the Ad5-eGFP-v5_KD negative controlvirus. Comparison with the diluted Ad5-eGFP-v5_KD infected samplesallowed to estimate the reduction in expression to be 2-fold. Equalloading of the 30 μg samples is demonstrated by immunodetection ofβ-actin after removal of the MAPKAPK5 antibody by a ‘strippingprocedure’ (5 minutes boiling of the membrane in PBST). Immunodetectionof β-actin is performed according to the method described for MAPKAPK5detection, but using a goat polyclonal antibody against β-actin (SantaCruz, Cat N^(o) SC-1615) at a 1000 fold dilution as primary antibody anda rabbit anti goat antibody at a 50000 fold dilution as a secondaryantibody. Results of this experiment are given in FIG. 4. Takentogether, this experiment demonstrated the functionality of the Ad-siRNAvirus produced to reduce the MAPKAPK5 expression levels in primary humanSFs.

(B) MAPKAPK5 Knock-Down Ad-siRNA Reduces SF-Induced MMP1 Expression

The efficacy of Ad5-MAPKAPK5-v13_KD virus in the ‘MMP assay’ is testedas follows. Day 1, SFs (passage 9 to 10) are seeded in 96 well plates ata density of 3000 cells per well in complete synovial growth medium(Cell Applications). One day later, the cells are infected withincreasing amounts (3, 6; 9, 12 or 15 μl) of following viruses:Ad5-eGFP-v5_KD, Ad5-MAPKAPK5-v12_KD, Ads-MAPKAPK5-v13_KD,Ad5-MMP1-v10_KD. The virus load is corrected by addition of the neutralvirus Ad5-Luc-v13_KD to bring the final virus volume on the cells to 15μL in every well. This correction guarantees that the effects observeddo not result from the virus load applied to the cells. The cells arethen incubated for 5 days before the activation step. This step involvesthe replacement, in every well, of the growth medium by 754 of M199medium supplemented with 25 μL of ‘complex trigger’. 48 hrs after theactivation step, the supernatant is collected and subjected to the MMP1ELISA as described in Example 1. The results of the experiment are shownin FIG. 5. The quality of the experiment is demonstrated by the efficacyof the Ad-siRNA virus targeting MMP1 itself. This positive control virusstrongly reduces the MMP1 expression by SFs, whereas the negativecontrol virus, designed to target the expression of luciferase, does notinfluence the levels of MMP1 expression. Two viruses used to validatethe MAPKAPK5 target (Ad5-MAPKAPK5-v12_KD and Ad5-MAPKAPK5-v13) do alsolead to a clear reduction of the complex trigger induced MMP1 expressionby primary human SFs. It can be concluded, from this experiment, thatMAPKAPK5 represents a valuable drug target that is shown to modulateMMP1 expression in SFs. Similarly, the inhibition of MAPKAPK5 enzymaticactivity by a small molecule compound is expected to reduce the ‘complexcytokine’ induced MMP1 expression in the ‘MMP assay’. The inhibition ofMAPKAPK5 enzymatic activity by a small molecule compound is alsopredicted to reduce the degradation of the joint associated with RA.

(C) In Vitro ‘MMP Assay’ Testing of Compounds Inhibiting MAPKAPK5

Compounds inhibiting the MAPKAPK5 activity in a biochemical assay (i.e.cell free, using purified enzyme), are tested in the ‘MMP assay’according to following protocol.

The compound master stocks (all at 10 mM concentration in 100% DMSO) arediluted 10-fold in water (Distilled water, GIBCO, DNAse and RNAse free)to obtain a 1 mM intermediate work stock in 10% DMSO. This intermediatework stock is further diluted either 3-fold (or 10-fold) in 10% DMSO toobtain an intermediate work stock of 333 μM (or 100 μM) concentration,respectively, in 10% DMSO. The 1 mM as well as 333 μM (or 100 μM)intermediate work stocks are then further diluted 10-fold in 1.1% DMSOto obtain the 10× workstocks at 100 μM and 33.3 μM (or 10 μM)concentration in 2% DMSO. This 10× work stock is then diluted 10-fold inM199 medium supplemented with 1% FBS to obtain the final ‘1× compoundpreparation’ containing the compounds at 10 μM and 3.33 μM (or 1 μM) aswell as 0.2% DMSO. These are the final conditions at which the compoundsare tested on the cells. In parallel, the 10× work stock is diluted10-fold in ‘complex trigger’ (i.e. the supernatant of TNF-α treated THP1cells produced as described in Example 1) that is diluted 2-fold in M199supplemented with 1% FBS to produce the ‘1× compound in 50% complextrigger preparation’.

At day 1, RASFs are seeded in 96 well plates (Flat bottom, tissueculture treated, Greiner) at a density of 3000 cells/well in completesynovial growth medium (Cell Applications). Day 5, the compounds areadded to the cultured cells as follows. Medium is completely removedfrom the cells and replaced by 75 μL of the ‘1× compound preparations’containing the compounds at either 10 μM or 3.33 μM (or 1 μM) in M199medium supplemented with 1% FBS and 0.2% DMSO. After an incubationperiod of 2 hours, which allows the compounds to equilibrate and enterthe cells; 25 μL of the ‘1× compound in 50% complex triggerpreparations’ are added to the wells on top of the 1× compoundpreparation', in the wells containing the corresponding compounds atcorresponding concentration. In this way, an 8-fold diluted complextrigger is ultimately applied to the cells. An incubation of 48 hrs isthen performed and 200 of the cell supernatant is then processed in theMMP1 ELISA as described above, delivering raw data (RLU: relativeluminescence units). Following controls are included in the experiments.A maximal signal control, in which the cells are activated by thecomplex trigger but only the 0.2% DMSO vehicle (and thus no compound) isadded. This control indicates the maximal level of MMP1 that can beachieved in the test. A minimal signal control is also included in theseexperiments. Here, cells are not triggered. The medium of the cells isthen changed to 100 μl M199 medium supplemented with 1% FBS at day 5.This control returns the basal MMP1 levels produced by the RASFs. Thepercent inhibition of the MMP1 expression achieved by the compounds isthen calculated based on the RLU data returned by the ELISA withfollowing formula: [[(maximal MMP1 levels−minimal MMP1 levels)−(MMP1level compound X at concentration Y−minimal MMP1 levels)]/(maximal MMP1levels−minimal MMP1 levels)]×100.

Toxicity of the compounds is assessed as follows. Day 1, SFs are seededin white, tissue culture treated 96 well plates at a density of 3000cells per well in 100 μL complete synovial growth medium. The compoundhandling, compound addition to the cells as well as activation of thecells is further performed as described above in this example for thedetermination of the MMP1 levels. After the 48 hrs incubation period,the medium is removed from the wells, replaced by 50 μL fresh M199medium supplemented with 1% FBS. 50 μL of substrate (Promega CelltiterGlow cell viability kit) is then added to the wells. After an incubationperiod of 10 min, luminescence signal is measured. A reduction of theluminescence signal by more than 50% as compared to the maximal controlwells is considered to reflect significant toxicity. No toxicity isobserved for the compounds tested in the ‘MMP assay’.

It should be understood that factors such as the differential cellpenetration capacity of the various compounds can contribute todiscrepancies between the activity of the compounds in the in vitrobiochemical and cellular MMP assays.

Example 3 Assay to Assess Effect of Compounds on Cytokine Release byHuman PBMCs

Human peripheral blood mononuclear cells (PBMCs) are isolated from“buffy coats” prepared from the blood of healthy volunteers, isolatedessentially according to method of Bøyum (1984). In brief, buffy coat isdiluted 1:1 with 1×PBS (Gibco) and 30 mL is carefully put on top of 20mL Lymphoprep™ (Lucron Bioproducts) in 50 mL Falcon tubes. Aftercentrifugation (35 min, 400 g, 18° C.) the mononuclear cells arecollected from the white interphase and washed 3 times with 1×PBS byresuspending and centrifugation (10 min, 200 g). Isolated PBMCs arefinally resuspended in RPMI 1640 (Cat. No. 21875, Gibco) that issupplemented with 10% heat-inactivated FBS (Hyclone).

For the assay PBMCs are seeded at 2.5E6 cells/mL in 160 μL in 96-wellplates (Nunc). Serial dilution of the test compounds are made first inDMSO (Sigma) and then diluted 50-fold in M199 medium (Gibco) containing1% heat-inactivated FBS. Compounds are further 1/10 diluted in the assayplates to obtain final DMSO concentration of 0.2%. Cells arepreincubated with the compounds for 1 hr at 37° C., 5% CO₂. Then, cellsare stimulated with LPS (Escherichia coli serotype 026:B6, Cat.No.L2654, Sigma) that is added in a volume of 20 μL to a finalconcentration of 1 μg/mL and cells are further cultured for 24 hr. Theplates are centrifuged and the supernatant is collected and stored at−80° C. until analysis of appropriate dilutions in ELISAs.

The following 384-well chemiluminescent ELISA protocol was developed tomeasure TNFα levels in the supernatant: White Lumitrac 600 384-wellplates (Greiner) are coated with (40 μL/well) anti-TNFα capture antibody(Cat. No. 551220, BD Pharmingen) that is diluted to 1 μg/mL in 1×PBS(Gibco). After overnight incubation at 4° C., plates are washed with1×PBS (80 g NaCl, 2 g KCl (Sigma), 11.5 g Na₂HPO₄.7H2O and 2 g KH₂PO₄ in10 L milliQ; pH 7.4) and blocked with 100 μL/well buffer B (1×PBScontaining 1% BSA (Sigma), 5% sucrose (Sigma) and 0.05% NaN₃ (Sigma)).After 4 hr incubation at RT, blocking buffer is removed and plates arewashed once with PBST (1×PBS with 0.05% Tween-20 (Sigma)). Then, 40 μLof sample is transferred to the ELISA plates and plates are incubated at4° C. The next day, plates are washed 3 times (twice with PBST and oncewith PBS) and 35 μL/well biotinylated anti-TNFα antibody (Cat. No.554511, BD Pharmingen) diluted first to a concentration of 250 ng/ml inbuffer D (1×PBS with 1% BSA) is added. After 2 h of incubation at RT,plates are washed as described above and 35 μL/well of a 1/2000 dilutionof streptavidin-HRP conjugate (Cat. No. SNN2004, Biosource) in buffer Dis added. After 45 min, plates are washed as described above andincubated for 5 min with 50 μl/well BM Chemiluminescence ELISA SubstratePOD (Roche). Readout is performed on the Luminoscan Ascent Luminometer(Labsystems) with an integration time of 100 msec delivering raw data(RLU: relative luminescence units). The following controls are includedin the experiments, a maximal signal control, in which the cells areactivated by LPS but only the 0.2% DMSO vehicle (and thus no compound)is added. This control indicates the maximal level of TNFα that can beachieved in the test. A minimal signal control is also included in theseexperiments. Here, cells are not triggered. This control returns thebasal TNFα levels produced by the PBMCs. The percent inhibition (PIN) ofthe TNFα release, achieved by the compounds is then calculated based onthe RLU data returned by the ELISA with following formula: 100−[((TNFαlevel compound X at concentration Y−minimal TNFα levels)/(maximal TNFαlevels−minimal TNFα levels))×100]. Where compounds are tested at 8concentrations (1/3 serial dilution), EC50-values can be calculated bycurve fitting of the means of the PIN data achieved for a compound ateach test concentration.

To assay the effect of compounds on the release of IL1 and IL6 by LPSstimulated PBMC cultures, appropriate dilutions of the supernatant canbe measured using the same ELISA protocol as described above. Matchedpair antibodies for IL1 and IL6 ELISA (all from R&D Systems) may be usedas follows: anti-IL1 capture antibody (Cat. No. MAB601) used at 0.5μg/mL, biotinylated anti-IL1 detection antibody (Cat. No. BAF201) usedat 50 ng/mL; anti-IL6 capture antibody (Cat. No. MAB206) used at 1μg/mL; biotinylated anti-IL6 detection antibody (Cat. No. BAF206) usedat 50 ng/mL.

For the purpose of Table 2 below, the MAPKAPK5 IC₅₀ of each compound,which can be determined using the assay method described herein, isexpressed as follows:

++++ compound exhibited MAPKAPK5 IC₅₀ 1-100 nM

+++ compound exhibited MAPKAPK5 IC₅₀ 101-500 nM

++ compound exhibited MAPKAPK5 IC₅₀ 501-1000 nM

+ compound exhibited MAPKAPK5 IC₅₀>1000 nM

N.A. No data available for compound

EX# NAME MAPKAPK5 1 5-(8-(4-(MORPHOLINOMETHYL)THIAZOL-2- +++YLAMINO)IMIDAZO[1,2-A]PYRAZIN-5- YL)ISOINDOLIN-1-ONE 25-(8-(5-(HYDROXYMETHYL)THIAZOL-2- N.A. YLAMINO)IMIDAZO[1,2-A]PYRAZIN-5-YL)ISOINDOLIN-1-ONE 3 N-(5-(1H-PYRAZOL-4-YL)- + [1,2,4]TRIAZOLO[1,5-A]PYRAZIN-8-YL)-4- (MORPHOLINOMETHYL)THIAZOL-2-AMINE 44-(8-(4-(PIPERIDIN-1-YLMETHYL)THIAZOL-2- ++++YLAMINO)-[1,2,4]TRIAZOLO[1,5-A]PYRAZIN- 5-YL)FURAN-2-CARBOXAMIDE 54-(8-(1-(2-MORPHOLINOETHYL)-1H- N.A. IMIDAZOL-2-YLAMINO)-[1,2,4]TRIAZOLO[1,5-A]PYRAZIN-5- YL)FURAN-2-CARBOXAMIDE 65-(8-(THIAZOL-2-YLAMINO)IMIDAZO[1,2- +++ A]PYRAZIN-5-YL)ISOINDOLIN-1-ONE7 N-(5-(1H-PYRAZOL-4-YL)IMIDAZO[1,2- + A]PYRAZIN-8-YL)THIAZOL-2-AMINE 85-(8-(5-(ETHYLSULFONYL)THIAZOL-2- N.A. YLAMINO)IMIDAZO[1,2-A]PYRAZIN-5-YL)ISOINDOLIN-1-ONE 9 5-(8-(4-(PIPERIDIN-1-YLMETHYL)THIAZOL-2- ++++YLAMINO)IMIDAZO[1,2-A]PYRAZIN-5- YL)ISOINDOLIN-1-ONE 10N,N,4-TRIMETHYL-2-(5-(1-OXOISOINDOLIN-5- +++ YL)IMIDAZO[1,2-A]PYRAZIN-8-YLAMINO)THIAZOLE-5-CARBOXAMIDE 11 5-(8-(5-METHYLTHIAZOL-2- ++YLAMINO)IMIDAZO[1,2-A]PYRAZIN-5- YL)ISOINDOLIN-1-ONE 125-(8-(5,6-DIHYDRO-4H- + CYCLOPENTA[D]THIAZOL-2-YLAMINO)IMIDAZO[1,2-A]PYRAZIN-5- YL)ISOINDOLIN-1-ONE 135-(8-(5-(MORPHOLINE-4- ++ CARBONYL)THIAZOL-2-YLAMINO)IMIDAZO[1,2-A]PYRAZIN-5- YL)ISOINDOLIN-1-ONE 145-(8-(5-(2-(PIPERIDIN-1-YL)ETHYL)-1,3,4- ++THIADIAZOL-2-YLAMINO)IMIDAZO[1,2- A]PYRAZIN-5-YL)ISOINDOLIN-1-ONE 15N-(5-(1H-PYRAZOL-4-YL)IMIDAZO[1,2- + A]PYRAZIN-8-YL)-5-(MORPHOLINOMETHYL)THIAZOL-2-AMINE 165-(8-(5-(MORPHOLINOMETHYL)THIAZOL-2- +++YLAMINO)IMIDAZO[1,2-A]PYRAZIN-5- YL)ISOINDOLIN-1-ONE 17N-(5-(1H-PYRAZOL-4-YL)IMIDAZO[1,2- + A]PYRAZIN-8-YL)-5-((DIMETHYLAMINO)METHYL)THIAZOL-2- AMINE 185-(8-(5-((4-ISOPROPYLPIPERAZIN-1- +++ YL)METHYL)THIAZOL-2-YLAMINO)IMIDAZO[1,2- A]PYRAZIN-5-YL)ISOINDOLIN-1-ONE 195-(8-(1,3,4-THIADIAZOL-2- + YLAMINO)IMIDAZO[1,2-A]PYRAZIN-5-YL)ISOINDOLIN-1-ONE 20 5-(8-(5-(4-METHYLPIPERAZIN-1-YL)-1,3,4- ++THIADIAZOL-2-YLAMINO)IMIDAZO[1,2- A]PYRAZIN-5-YL)ISOINDOLIN-1-ONE 21(2-(5-(1H-PYRAZOL-4-YL)IMIDAZO[1,2- N.A. A]PYRAZIN-8-YLAMINO)THIAZOL-5-YL)(MORPHOLINO)METHANONE 22 (2-(5-(1H-PYRAZOL-4-YL)IMIDAZO[1,2- N.A.A]PYRAZIN-8-YLAMINO)THIAZOL-5- YL)(PIPERIDIN-1-YL)METHANONE 23(2-(5-(1H-PYRAZOL-4-YL)IMIDAZO[1,2- +A]PYRAZIN-8-YLAMINO)THIAZOL-5-YL)(4- ISOPROPYLPIPERAZIN-1-YL)METHANONE24 5-(8-(5-(PIPERIDIN-1-YLMETHYL)THIAZOL-2- +YLAMINO)IMIDAZO[1,2-A]PYRAZIN-5- YL)ISOINDOLIN-1-ONE 25N-(5-(1H-PYRAZOL-4-YL)- + [1,2,4]TRIAZOLO[1,5-A]PYRAZIN-8-YL)-4-(PIPERIDIN-1- YLMETHYL)THIAZOL-2-AMINE

The present invention relates also to a method of treatment orprevention of inflammatory diseases, which comprises administering to asubject in need thereof, a therapeutically effective inhibitor ofMitogen-Activated Protein Kinase-Activated Protein Kinase 5 inhibitingamount of a compound according to Formula I.

Another aspect of the present method invention relates to a method oftreatment or prophylaxis of a condition characterised by abnormal matrixmetallo proteinase activity, which comprises administering atherapeutically effective amount of a matrix metallo proteinaseinhibiting compound according to Formula I.

A further aspect of the present method invention is a method oftreatment or prophylaxis of a condition selected from diseases involvingdegradation of extra-cellular matrix, which comprises administering atherapeutically effective matrix metallo proteinase inhibiting amount ofa compound according to Formula I.

A yet further aspect of the present method invention is a method oftreatment or prophylaxis of a condition selected from diseases involvingabnormal cellular expression of MMP1, which comprises administering atherapeutically effective matrix metallo proteinase inhibiting amount ofa compound according to Formula I.

A special embodiment of the present method invention is a method oftreatment or prevention of rheumatoid arthritis, which comprisesadministering to a subject in need thereof, a therapeutically effectiveamount of a compound according to Formula I.

This invention also relates to the use of the present compounds in themanufacture of a medicament for treatment or prophylaxis of a conditionprevented, ameliorated or eliminated by administration of an inhibitorof Mitogen-Activated Protein Kinase-Activated Protein Kinase 5, or acondition characterised by abnormal collagenase activity, or a conditionselected from diseases involving inflammation, most preferably in forthe treatment of rheumatoid arthritis.

Administering of the compound of the present invention to the subjectpatient includes both self-administration and administration by anotherperson. The patient may be in need of treatment for an existing diseaseor medical condition, or may desire prophylactic treatment to prevent orreduce the risk for diseases and medical conditions affected by adisturbance in bone metabolism. The compound of the present inventionmay be delivered to the subject patient orally, transdermally, viainhalation, injection, nasally, rectally or via a sustained releaseformulation.

A preferred regimen of the present method comprises the administrationto a subject in suffering from a disease condition characterized byinflammatory, with an effective matrix metallo-protease inhibitingamount of a compound of the present invention for a period of timesufficient to reduce the abnormal levels of extracellular matrixdegradation in the patient, and preferably terminate, theself-perpetuating processes responsible for said degradation. A specialembodiment of the method comprises administering of an effective matrixmetallo-protease inhibiting amount of a compound of the presentinvention to a subject patient suffering from or susceptible to thedevelopment of rheumatoid arthritis, for a period of time sufficient toreduce or prevent, respectively, collagen and bone degradation in thejoints of said patient, and preferably terminate, the self-perpetuatingprocesses responsible for said degradation.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

A preferred therapeutically effective amount of the compound of thepresent invention to administer to a subject patient is about 0.1 mg/kgto about 10 mg/kg administered from once to three times a day. Forexample, an effective regimen of the present method may administer about5 mg to about 1000 mg of said compound of the present invention fromonce to three times a day. It will be understood, however, that thespecific dose level for any particular subject patient will depend upona variety of factors including the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination and the severity of the particularinflammatory condition. A consideration of these factors is well withinthe purview of the ordinarily skilled clinician for the purpose ofdetermining the therapeutically effective or prophylactically effectivedosage amount needed to prevent, counter, or arrest the progress of thecondition.

Compounds of the invention can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise at least one compound of the invention and at least onepharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include solidcarriers such as lactose, magnesium stearate, terra alba, sucrose, talc,stearic acid, gelatin, agar, pectin, acacia or the like; and liquidssuch as vegetable oils, arachis oil and sterile water, or the like, anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. This listing ofpharmaceutically acceptable carriers is not to be construed as limiting.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum mono stearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a compound according to an embodiment of the invention)in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

A compound according to an embodiment of the invention may be providedas a salt, preferably as a pharmaceutically acceptable salt of compoundsof formula I. Examples of pharmaceutically acceptable salts of thesecompounds include those derived from organic acids such as acetic acid,malic acid, tartaric acid, citric acid, lactic acid, oxalic acid,succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid,phenylacetic acid, mandelic acid, methanesulphonic acid,benzenesulphonic acid and p-toluenesulphonic acid, mineral acids such ashydrochloric and sulphuric acid and the like, giving methanesulphonate,benzenesulphonate, p-toluenesulphonate, hydrochloride and sulphate, andthe like, respectively or those derived from bases such as organic andinorganic bases. Examples of suitable inorganic bases for the formationof salts of compounds for this invention include the hydroxides,carbonates, and bicarbonates of ammonia, lithium, sodium, calcium,potassium, aluminium, iron, magnesium, zinc and the like. Salts can alsobe formed with suitable organic bases. Such bases suitable for theformation of pharmaceutically acceptable base addition salts withcompounds of the present invention include organic bases which arenontoxic and strong enough to form salts. Such organic bases are alreadywell known in the art and may include amino acids such as arginine andlysine, mono-, di-, or trihydroxyalkylamines such as mono-, di-, andtriethanolamine, choline, mono-, di-, and trialkylamines, such asmethylamine, dimethylamine, and trimethylamine, guanidine;N-methylglucosamine; N-methylpiperazine; morpholine; ethylenediamine;N-benzylphenethylamine; tris(hydroxymethyl)aminomethane; and the like.

Salts of compounds according to an embodiment of the invention may beprepared in a conventional manner using methods well known in the art.Acid addition salts of said basic compounds may be prepared bydissolving the free base compounds according to the first or secondaspects of the invention in aqueous or aqueous alcohol solution or othersuitable solvents containing the required acid. Where a compound of theinvention contains an acidic function, a base salt of said compound maybe prepared by reacting said compound with a suitable base. The acid orbase salt may separate directly or can be obtained by concentrating thesolution e.g. by evaporation. The compounds of this invention may alsoexist in solvated or hydrated forms.

It will be appreciated by those skilled in the art that the foregoingdescription is exemplary and explanatory in nature, and is intended toillustrate the invention and its preferred embodiments. Through routineexperimentation, an artisan will recognise apparent modifications andvariations that may be made without departing from the spirit of theinvention. Thus, the invention is intended to be defined not by theabove description, but by the following claims and their equivalents.

REFERENCES

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From the foregoing description, various modifications and changes in thecompositions and methods of this invention will occur to those skilledin the art. All such modifications coming within the scope of theappended claims are intended to be included therein.

It should be understood that factors such as the differential cellpenetration capacity of the various compounds can contribute todiscrepancies between the activity of the compounds in the in vitrobiochemical and cellular assays.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

At least some of the chemical names of compounds of the invention asgiven and set forth in this application, may have been generated on anautomated basis by use of a commercially available chemical namingsoftware program, and have not been independently verified.Representative programs performing this function include the Lexichemnaming tool sold by Open Eye Software, Inc. and the Autonom Softwaretool sold by MDL, Inc. In the instance where the indicated chemical nameand the depicted structure differ, the depicted structure will control.

Chemical structures shown herein were prepared using either ChemDraw® orISIS®/DRAW. Any open valency appearing on a carbon, oxygen or nitrogenatom in the structures herein indicates the presence of a hydrogen atom.Where a chiral center exists in a structure but no specificstereochemistry is shown for the chiral center, both enantiomersassociated with the chiral structure are encompassed by the structure.

1. A compound according to formula I:

wherein Z is N; R¹ is H, or substituted or unsubstituted C₁-C₆ alkyl; R²is H, C₁-C₆ alkyl, C₃-C₇ cycloalkyl or C₃-C₇ cycloalkyl-C₁-C₆ alkyl,optionally substituted with one or more of F and Cl; R⁸ is substitutedor unsubstituted heteroaryl; R⁹ is selected from substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl; or apharmaceutically acceptable salt, solvate or prodrug thereof; andstereoisomers and tautomers thereof.
 2. The compound according to claim1 wherein R⁸ is substituted or unsubstituted 5-membered heteroaryl. 3.The compound according to claim 2 wherein R⁸ is selected fromsubstituted or unsubstituted pyrrolyl, furanyl, thienyl, pyrazolyl,oxazolyl, imidazolyl, thiazolyl, 1,3,4-thiadiazolyl, and1,2,4-thiadiazolyl.
 4. The compound according to claim 3 wherein R⁸ isselected from substituted pyrrolyl, furanyl, thienyl, pyrazolyl,oxazolyl, imidazolyl, thiazolyl, 1,3,4-thiadiazolyl, and1,2,4-thiadiazolyl and the substitution is selected from C₁-C₆ alkyl,substituted C₁-C₆ alkyl, carbamoyl, C₁-C₆ hydroxyalkyl, C₁-C₆ haloalkyl,C₁-C₆ alkoxy, cyano, amino, sulfo, aryl, C₃-C₇ cycloalkyl, aralkyl,3-7-membered heterocycloalkyl and heteroaryl.
 5. The compound accordingto claim 4 wherein R⁸ is selected from substituted pyrrolyl, furanyl,thienyl, pyrazolyl, oxazolyl, imidazolyl, thiazolyl, 1,3,4-thiadiazolyl,and 1,2,4-thiadiazolyl and the substitution is selected from is Me,i-Pr, cyclopropyl, CH₂OH, CONH₂, CONMe₂, NMe₂, NEt₂, SO₂Me, or SO₂Et. 6.The compound according to claim 1 wherein R⁸ is selected fromsubstituted pyrrolyl, furanyl, thienyl, pyrazolyl, oxazolyl, imidazolyl,thiazolyl, 1,3,4-thiadiazolyl, and 1,2,4-thiadiazolyl and thesubstitution is -L-R^(8d); wherein L is selected from bond, alkylene,heteroalkylene, —O—, —N(R^(8e))—, —CO—, —CO₂—, —SO—, —SO₂—,—CON(R^(8e))—, —SO₂N(R^(8e))—, —N(R^(8e))CO—, —N(R^(8e))SO₂—,—N(R^(8e))CO N(R^(8e))—, —N(R^(8e))SO₂N(R^(8e))—; and R^(8d) is selectedfrom substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted C₃-C₇ cycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted 3-7-membered heterocycloalkyl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted amino,substituted or unsubstituted aralkyl, substituted or unsubstitutedheteroarylalkyl and substituted or unsubstituted aminoalkyl; and R^(8e)is selected from H, substituted or unsubstituted C₁-C₆ alkyl andsubstituted or unsubstituted C₃-C₇ cycloalkyl.
 7. The compound accordingto claim 6, wherein R⁸ is

wherein L, and R^(8d) are as in claim 6; and R^(8c) is independentlyselected from hydrogen, and substituted or unsubstituted C₁-C₆ alkyl. 8.The compound according to claim 7, wherein L is —CON(R^(8e))— orSO₂N(R^(8e))—; R^(8d) is selected from substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted C₃-C₇ cycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted 3-7-memberedheterocycloalkyl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted aralkyl, substituted or unsubstituted heteroarylalkyland substituted or unsubstituted aminoalkyl; and R^(8e) is selected fromH, substituted or unsubstituted C₁-C₆ alkyl.
 9. The compound accordingto claim 7, wherein L is —CONH— or SO₂NH—; and R^(8d) is selected fromH, C₁-C₆ alkylaminoethyl, diC₁-C₆ dialkylaminoethyl, C₃-C₇ cycloalkyl,heterocycloalkyl, arylalkyl, and heteroarylalkyl.
 10. The compoundaccording to claim 9, wherein L is —CONH— or SO₂NH—; and R^(8d) isselected from H, methylaminoethyl, ethylaminoethyl, dimethylaminoethyl,diethylaminoethyl, substituted or unsubstituted pyrrolidinyl, benzyl andpyridylmethyl.
 11. The compound according to claim 7, wherein L is bond,—CO—, SO₂, —(CH₂)_(m1)—, —O(CH₂)_(m1)—, —NH(CH₂)_(m1)—,—CON(H)(CH₂)_(m1)—, or —SO₂NH(CH₂)_(m1)—; the subscript m1 is selectedfrom 1-4; and R^(8d) is

and wherein the ring P is substituted or unsubstituted heterocycloalkyl.12. A compound according to formula IIa, IIb, IIc, IId, IIe, or IIf:

and wherein Z is N, L is bond, —CO—, SO₂, —(CH₂)_(m1)—, —O(CH₂)_(m1)—,—NH(CH₂)_(m1)—, —CON(H)(CH₂)_(m1)—, or —SO₂NH(CH₂)_(m1)—; the subscriptm1 is selected from 1-4, R^(8c) is independently selected from hydrogen,and substituted or unsubstituted C₁-C₆ alkyl, and the ring P issubstituted or unsubstituted heterocycloalkyl; and R⁹ is independentlyselected from substituted or unsubstituted aryl and heteroaryl; or apharmaceutically acceptable salt, solvate or prodrug thereof; andstereoisomers and tautomers thereof.
 13. The compound according to claim12, wherein L is a bond.
 14. The compound according to claim 12, whereinL is —CO—.
 15. The compound according to claim 12, wherein L is —SO₂—.16. The compound according to claim 12, wherein L is —CON(H)—CH₂—CH₂—,or —SO₂NH—CH₂—CH₂—.
 17. The compound according to claim 12, wherein L is—OCH₂—CH₂— or —NHCH₂—CH₂—.
 18. The compound according to claim 12,wherein the ring P is substituted or unsubstituted piperidine,morpholine or piperazine.
 19. The compound according to claim 12,wherein the ring P is substituted or unsubstituted:

and R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl.
 20. The compoundaccording to claim 19 wherein R^(8b) is H, Me, Et, Pr, i-Pr, t-Bu,CH₂CONH₂, cyclopropyl or cyclopropylmethyl.
 21. The compound accordingto claim 1 wherein the compound is according to formula IIIa, IIIb,IIIc, IIId, IIIe, IIIf, IIIg, IIIh, IIIi, IIIj, IIIk, IIIl, or IIIm:

and wherein L is bond, —CO—, SO₂, —(CH₂)_(m1)—, —O(CH₂)_(m1)—,—NH(CH₂)_(m1)—, —CON(H)(CH₂)_(m1)—, or —SO₂NH(CH₂)_(m1)—; the subscriptm1 is selected from 1-4; R^(8b) is hydrogen, substituted orunsubstituted C₁-C₆ alkyl or substituted or unsubstituted C₃-C₇cycloalkyl; and R⁹ is independently selected from substituted orunsubstituted aryl and heteroaryl; or a pharmaceutically acceptablesalt, solvate or prodrug thereof; and stereoisomers and tautomersthereof.
 22. The compound according to claim 1 wherein the compound isaccording to formula IIIn, IIIo, IIIp, IIIq, IIIs, IIIt, IIIu, IIIv,IIIw, IIIx, or IIIy:

and wherein L is bond, —CO—, SO₂, —(CH₂)_(m1)—, —O(CH₂)_(m1)—,—NH(CH₂)_(m1)—, —CON(H)(CH₂)_(m1)—, or —SO₂NH(CH₂)_(m1)—; the subscriptm1 is selected from 1-4; R^(8b) is hydrogen, substituted orunsubstituted C₁-C₆ alkyl or substituted or unsubstituted C₃-C₇cycloalkyl; and R⁹ is independently selected from substituted orunsubstituted aryl and heteroaryl; or a pharmaceutically acceptablesalt, solvate or prodrug thereof; and stereoisomers and tautomersthereof.
 23. The compound according to claim 21, wherein L is a bond.24. The compound according to claim 21, wherein L is —CH₂—.
 25. Thecompound according to claim 21, wherein L is —CO—.
 26. The compoundaccording to claim 21, wherein L is —SO₂—.
 27. The compound according toclaim 21, wherein L is —CON(H)—CH₂—CH₂—, or —SO₂NH—CH₂—CH₂—.
 28. Thecompound according to claim 21, wherein L is —OCH₂—CH₂— or —NHCH₂—CH₂—.29. The compound according to claim 21, wherein R^(8b) is H, Me, Et, Pr,i-Pr, t-Bu, i-Bu, cyclopropyl or cyclopropylmethyl.
 30. The compoundaccording to claim 1, wherein R⁹ is selected from substituted orunsubstituted phenyl.
 31. The compound according to claim 1, wherein R⁹is selected from substituted or unsubstituted pyridyl, indolyl,isoindolyl, pyrrolyl, furanyl, thienyl, pyrazolyl, oxazolyl, andthiazolyl.
 32. The compound according to claim 1, wherein R⁹ is

and each of A¹, A² and A³ is independently selected from S, O, N,NR^(9a), and CR^(9a); each of R^(9a) is independently H or substitutedor unsubstituted C₁-C₆ alkyl; and R^(9b) is CONH₂, CONHMe, or CN. 33.The compound according to claim 1, wherein R⁹ is


34. The compound according to claim 1, wherein R⁹ is


35. The compound according to claim 1, wherein R⁹ is

and wherein the subscript m is selected from 0-4 and each R^(9d) isindependently substituted or unsubstituted C₁-C₆ alkyl or halo.
 36. Thecompound according to claim 1, wherein R⁹ is

and wherein the subscript m is selected from 0-4 and each R^(9d) isindependently substituted or unsubstituted C₁-C₆ alkyl or halo.
 37. Thecompound according to claim 1, wherein R⁹ is

wherein the subscript m is selected from 0-3 and each R^(9d) isindependently substituted or unsubstituted C₁-C₆ alkyl or halo.
 38. Thecompound according to claim 35, wherein m is 1 or 2 and each R^(9d) isMe, Cl or F.
 39. The compound according to claim 1 wherein the compoundis according to formula IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVi,IVj, IVk or IVl:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers andtautomers thereof.
 40. The compound according to claim 1 wherein thecompound is according to formula IVm, IVn, IVo, IVp, IVq, IVr, IVs, IVt,IVu, IVv, IVw or IVx:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers andtautomers thereof.
 41. The compound according to claim 1 wherein thecompound is according to formula Va, Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj,Vk or Vl:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; and R^(9e) is hydrogen,Me, or CN; or a pharmaceutically acceptable salt, solvate or prodrugthereof; and stereoisomers and tautomers thereof.
 42. The compoundaccording to claim 1 wherein the compound is according to formula Vm,Vn, Vo, Vp, Vq, Vr, Vs, Vt, Vu, Vv, Vw or Vx:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; and R^(9e) is hydrogen,Me, or CN; or a pharmaceutically acceptable salt, solvate or prodrugthereof; and stereoisomers and tautomers thereof.
 43. The compoundaccording to claim 1 wherein the compound is according to formula VIa,VIb, VIc, VId, VIe, VIf, VIg, VIh, VIi, VIj, VIk or VIl:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers andtautomers thereof.
 44. The compound according to claim 1 wherein thecompound is according to formula VIm, VIn, VIo, VIp, VIq, VIr, VIs, VIt,VIu, VIv, VIw or VIx:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers andtautomers thereof.
 45. The compound according to claim 1 wherein thecompound is according to formula VIIa, VIIb, VIIc, VIId, VIIe, VIIf,VIIg, VIIh, VIIi, VIIj, VIIk or VIIl:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers andtautomers thereof.
 46. The compound according to claim 1 wherein thecompound is according to formula VIIm, VIIn, VIIo, VIIp, VIIq, VIIr,VIIs, VIIt, VIIu, VIIv, VIIw or VIIx:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers andtautomers thereof.
 47. The compound according to claim 1 wherein thecompound is according to formula VIIIa, VIIIb, VIIIc, VIIId, VIIIe,VIII, VIIIg, VIIIh, VIIIi, VIIIj, VIIIk or VIIIl:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; and R^(9e) is hydrogen,Me, or CN; or a pharmaceutically acceptable salt, solvate or prodrugthereof; and stereoisomers and tautomers thereof.
 48. The compoundaccording to claim 1 wherein the compound is according to formula VIIIm,VIIIn, VIIIo, VIIIp, VIIIq, VIIIr, VIIIs, VIIIt, VIIIu, VIIIv, VIIIw orVIIIx:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; and R^(9e) is hydrogen,Me, or CN; or a pharmaceutically acceptable salt, solvate or prodrugthereof; and stereoisomers and tautomers thereof.
 49. The compoundaccording to claim 1 wherein the compound is according to formula IXa,IXb, IXc, IXd, IXe, IXf, IXg, IXh, IXi, IXj, IXk or IXl:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers andtautomers thereof.
 50. The compound according to claim 1 wherein thecompound is according to formula IXm, IXn, IXo, IXp, IXq, IXr, IXs, IXt,IXu, IXv, IXw or IXx:

wherein R^(8b) is hydrogen, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₃-C₇ cycloalkyl; or a pharmaceuticallyacceptable salt, solvate or prodrug thereof; and stereoisomers andtautomers thereof.
 51. The compound according to claim 39, whereinR^(8b) is H.
 52. The compound according to claim 39, wherein R^(8b) isC₃-C₇ cycloalkyl.
 53. The compound according to claim 52, wherein R^(8b)is cyclopropyl.
 54. The compound according to claim 39, wherein R^(8b)is substituted or unsubstituted C₁-C₆ alkyl.
 55. The compound accordingto claim 39, wherein R^(8b) is Me, Et, Pr, i-Pr, t-Bu, i-Bu, CF₃,CH₂CF₃, or cyclopropylmethyl.
 56. The compound according to claim 1wherein the compound is according to formula Xa, Xb or Xc:

wherein R^(8c) is hydrogen, or substituted or unsubstituted C₁-C₆ alkyl.57. The compound according to claim 1 wherein the compound is accordingto formula Xd, Xe or Xf:

wherein R^(8c) is hydrogen, or substituted or unsubstituted C₁-C₆ alkyl;and R^(9e) is hydrogen, Me, or CN; or a pharmaceutically acceptablesalt, solvate or prodrug thereof; and stereoisomers and tautomersthereof.
 58. The compound according to claim 1 wherein the compound isaccording to formula Xg, Xh or Xi:

and wherein R^(8c) is hydrogen, or substituted or unsubstituted C₁-C₆alkyl; or a pharmaceutically acceptable salt, solvate or prodrugthereof; and stereoisomers and tautomers thereof.
 59. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and apharmaceutically effective amount of a compound according to claim 1.60. The pharmaceutical composition of claim 59, wherein the carrier is aparenteral carrier.
 61. The pharmaceutical composition of claim 59,wherein the carrier is an oral carrier.
 62. The pharmaceuticalcomposition of claim 59, wherein the carrier is a topical carrier.